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Mitochondrial sorting and assembly machinery operates by β-barrel switching

Abstract

The mitochondrial outer membrane contains so-called β-barrel proteins, which allow communication between the cytosol and the mitochondrial interior1,2,3. Insertion of β-barrel proteins into the outer membrane is mediated by the multisubunit mitochondrial sorting and assembly machinery (SAM, also known as TOB)4,5,6. Here we use cryo-electron microscopy to determine the structures of two different forms of the yeast SAM complex at a resolution of 2.8–3.2 Å. The dimeric complex contains two copies of the β-barrel channel protein Sam50—Sam50a and Sam50b—with partially open lateral gates. The peripheral membrane proteins Sam35 and Sam37 cap the Sam50 channels from the cytosolic side, and are crucial for the structural and functional integrity of the dimeric complex. In the second complex, Sam50b is replaced by the β-barrel protein Mdm10. In cooperation with Sam50a, Sam37 recruits and traps Mdm10 by penetrating the interior of its laterally closed β-barrel from the cytosolic side. The substrate-loaded SAM complex contains one each of Sam50, Sam35 and Sam37, but neither Mdm10 nor a second Sam50, suggesting that Mdm10 and Sam50b function as placeholders for a β-barrel substrate released from Sam50a. Our proposed mechanism for dynamic switching of β-barrel subunits and substrate explains how entire precursor proteins can fold in association with the mitochondrial machinery for β-barrel assembly.

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Fig. 1: Structure of the SAMdimer complex.
Fig. 2: Subunit interactions in the SAMdimer complex.
Fig. 3: Structure of the SAMMdm10 complex.
Fig. 4: Dynamic assembly of the SAM complex and assembly of β-barrel proteins.

Data availability

Our cryo-EM density maps have been deposited in the Electron Microscopy Data Bank (https://www.ebi.ac.uk/pdbe/emdb/) under accession codes 30189, 30190 and 30191. Atomic coordinates have been deposited in the Protein Data Bank (https://www.rcsb.org) under accession codes 7BTW, 7BTX and 7BTY. Uncropped versions of all blots, gels and plates can be found in Supplementary Fig. 2Source data are provided with this paper.

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Acknowledgements

We thank the members of the Endo and Pfanner laboratories for discussion and comments on the manuscript; and N. Zufall for expert technical assistance. This work was supported by JSPS KAKENHI grants to T.E. (grant numbers 15H05705 and 2222703), K.I. (16K21680 and 18K11543) and H.T. (18K14640); a JST CREST grant to T.E (JPMJCR12M1); Deutsche Forschungsgemeinschaft (DFG) grants to T.B. (BE 4679/2-2; SFB 1218 project identification 269925409), N.P. (PF 202/9-1) and N.W. (WI 4506/1-1; SFB 1381 project identification 403222702), and, under Germany’s Excellence Strategy/Initiative, to T.B., N.P. and N.W. (CIBSS-EXC2189 project identification 390939984; GSC-4 Spemann Graduate School); and a European Research Council (ERC) Consolidator grant to N.W. (648235). We also acknowledge grants from the Takeda Science Foundation (to T.E.); and the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from the Japan Agency for Medical Research and Development (AMED) under grant numbers JP19am01011115 (to M.K.), JP19am0101114 (to K.I.), JP19am0101083 (to T.M.) and JP19am0101110 (to K.T). H.T. was supported by a Research Fellowship for Young Scientists from the Japan Society for the Promotion of Science (18J00358).

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Authors and Affiliations

Authors

Contributions

T.E. designed the research and wrote the paper. H.T. performed most of the experiments and wrote the paper. A.T. performed and M.K. supervised the cryo-EM measurements and data processing, including single-particle analyses. T.N. and O.N. supervised grid preparation, data processing and molecular modelling. C.L., J.V.B., L.-S.W., L.E., S.P.S., W.M. and J.Q. characterized yeast mutants and studied the organization of SAM complexes and interactions with precursor proteins. Y.Y. and K.T. performed comparative modelling and molecular dynamics flexible fitting for model building. N.P., N.W. and T.B. designed and wrote part of the paper. K.I. performed bioinformatics analyses and wrote part of the paper. J.S. assisted with sample preparation, and S.O. and T.M assisted with grid preparation for cryo-EM measurements. All authors contributed to the analysis and discussion of the results.

Corresponding author

Correspondence to Toshiya Endo.

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Peer review information Nature thanks Doron Rapaport, Gunnar von Heijne and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 Purification of SAM complexes.

a, Elution profile obtained by gel filtration of the purified SAMdimer complex in 0.02% GDN. b, c, SDS–PAGE gel for the SAMdimer complex stained with Coomassie blue (CBB) (b); the bands for Sam50, Sam35 and Sam37 were assigned by western blotting with antibodies (α) against each subunit (c). d, BN-PAGE gel for the SAMdimer complex stained with CBB. e, Elution profile obtained by gel filtration of the purified SAMMdm10 complex in 0.02% GDN. f, SDS–PAGE gel for the SAMMdm10 complex stained with CBB. g, BN-PAGE gel for the SAMMdm10 complex stained with CBB. Data are representative of two independent experiments (bd, f, g).

Extended Data Fig. 2 Cryo-EM analyses of the SAM complex.

Shown are data for the SAMdimer complex in GDN micelles (left), the SAMMdm10 complex in GDN micelles (central), and the SAMMdm10 complex in nanodiscs (right). a, Representative cryo-EM micrograph images of the complexes. b, Selected 2D classes showing various orientations. c, Workflows, showing the steps involved in data processing leading to the final structures of the SAM complexes. d, Left, local map resolutions of the final structures, with colours according to the scales, and right, FSC curves between the cryo-EM density maps and the atomic models of the SAM complexes.

Extended Data Fig. 3 Secondary structures and intersubunit interactions for Sam50a, Sam50b, Sam35, Sam37 and Mdm10.

af, Secondary structures and topology diagrams for Sam50a (a), Sam50b (b), Sam37 (c), Sam35 (d), Sam50a (e) and Mdm10 (f) are shown on the basis of the determined cryo-EM structures of the SAMdimer complex (ad) and the SAMMdm10 complex in nanodiscs (cf). Arrows, β-strands; rectangles, α-helices. Sam35 and Sam37 have GST-like folds63,64. Residues involved in intersubunit interactions are shown with circles plus residue numbers in colours that correspond to their interacting subunits; blue, Sam50a; light blue, Sam50b; orange, Sam35; yellow, Sam37; purple, Mdm10. Residues 171–191 of Sam37 were not sufficiently visible in the cryo-EM density map, and could be modelled only by molecular dynamics flexible fitting (see Methods). Other regions whose structures could not be determined owing to low cryo-EM densities are shown in Supplementary Fig. 1.

Extended Data Fig. 4 The hydrophobic belt and intrasubunit and intersubunit interactions of the SAMdimer complex.

ac, Close-up views of interactions within Sam50a. Interactions among β-strands 1, 14, 15 and 16 (a), among loop 1, loop 6 and β-strand 16 (b), and among loop 6 and β-strands 13–16 (c) are shown. β-Strands 1, 14, 15 and 16 exhibit hydrophobic interactions, and loop 6 also penetrates into the pore interior, forming salt bridges involving R366 (the functionally essential residue in the IRGF motif) and hydrogen bonds with β-strands 12–16. df, Close-up views of the interactions between the Sam50a and Sam50b barrels; the cryo-EM densities arising from two phospholipid-like molecules (with two acyl chains each) are shown in purple (e, f). Phospholipids including phosphatidylcholine and phosphatidylethanolamine have been shown to affect SAM activity and β-barrel biogenesis65,66,67, supporting a role of phospholipids at the Sam50 dimer interface in the functional integrity of SAM. gi, Comparison of the hydrophobic patches on the outer walls of Sam50a (g), the closed conformation of BamA (h) (PDB accession number 5D0O; https://doi.org/10.2210/pdb5D0O/pdb), and the open conformation of BamA (i) (PDB accession number 5LJO; https://doi.org/10.2210/pdb5LJO/pdb). In the closed conformation of BamA, a hydrophobic patch generated by F428, I430, Y432, V438, F440 and A442 is found around β-strands 1 and 2. By contrast, the hydrophobic patch is lost around the opening in Sam50a. Although hydrophobic residues (F131, A137 and A139) are also present in β-strands 1 and 2 of Sam50a, they may not form a stable hydrophobic patch owing to the loose conformation of β-strands 1 and 2, resulting from the loss of hydrogen bonds between β-strands 1 and 16. In the open conformation of BamA, β-strands 1 and 2 also become relatively loose, yet the hydrophobic patch is still maintained because there are more bulky hydrophobic residues than in Sam50a. jl, Intersubunit interactions are shown for Sam50a and Sam35.

Extended Data Fig. 5 Characterization of the SAMdimer complex.

a, b, Sam50 with an N-terminally attached His6 tag (His–Sam50) and/or Sam50 with an N-terminally attached 3HA tag (HA–Sam50) was expressed from the SAM50 or SAM37 promoter in sam50Δ yeast cells at levels nearly those of wild-type Sam50. Copy numbers of the plasmid-borne genes for the Sam50 derivatives are indicated at the top of each panel. a, Proteins of isolated mitochondria were analysed by SDS–PAGE and immunoblotting with the indicated antibodies; relative amounts of His–Sam50 were quantified and indicated below the relevant gel. b, Mitochondria were solubilized with digitonin and subjected to pull-down using Ni-NTA resin. Load (L), 5%; wash (W), 5%; eluate (E), 100%. Data are representative of two independent experiments. c, Bar diagrams showing the absolute copy numbers per yeast cell of SAM subunits and the major SAM substrates porin (Por1) and Tom40, based on quantifications of the yeast mitochondrial proteome under three different growth conditions30. d, e, Structural comparison of the laterally open (d) and closed (e) states of Sam50a and Sam50b in the SAMdimer complex. The laterally open structures represent the cryo-EM structure determined here, and the closed structures were modelled on the basis of the crystal structure of BamA (PDB accession number 4K3C; https://doi.org/10.2210/pdb4K3C/pdb). The side-chain carbon atoms of L481 are shown in space-filling form. The numerical values indicate the distances between the two L481 residues. The partially open lateral gates are roughly 21 Å apart, whereas the closed lateral gates are roughly 8 Å apart. f, Mitochondria containing Sam50C481 (C481, with a single cysteine in β-strand 16, exposing the SH group on the outside of the barrel)21 were incubated with the SH-specific homo-bifunctional crosslinking reagent BMOE (spacer length of 8 Å) for 30 min on ice. Protein complexes were solubilized with digitonin buffer and analysed by BN-PAGE followed by second dimension SDS–PAGE, western blotting and immunodecoration with the indicated antibodies. Sam50dimer, Sam50XL within the SAMdimer complex. Data are representative of three independent experiments.

Extended Data Fig. 6 Growth defects of sam35 and sam37 mutant strains.

a, Growth phenotypes of the indicated sam35 yeast mutants in the sam35Δ or sam35Δsam37Δ background on 5-FOA medium at 30 °C. Data are representative of two independent experiments. b, Growth phenotypes of the indicated sam37 yeast mutants on YPG medium at 36 °C. YPH499 is a wild-type yeast strain. Data are representative of three (left plate) or two (middle and right plates) independent experiments. c, d, Summary of the Sam35 and Sam37 constructs and mutant growth phenotypes shown in a, b. Green, viable; red (with dagger symbol), lethal; red (with minus symbol), no growth on YPG at 36 °C. e, Chemical amounts of Sam37 were imported into isolated wild-type (WT) and sam37Δ mitochondria. Mitochondria were lysed with digitonin, and protein complexes were analysed by BN-PAGE and immunodetection. SAM*, SAMdimer and SAMMdm10. Data are representative of three independent experiments. f, Isolated wild-type and sam35-15 mitochondria were subjected to heat treatment at 37 °C for 15–25 min, then analysed by BN-PAGE and immunodetection6,20. Data are representative of two independent experiments. As SAM35 (like SAM50) is essential for cell viability6,11,12,20,68, we used the temperature-sensitive sam35-15 yeast mutant. To minimize indirect effects, cells were grown at a permissive temperature and the isolated mitochondria were subjected to a heat treatment at a non-permissive temperature (37 °C). g, Isolated wild-type and sam35-15 mitochondria were heat-treated as in f, then incubated with a radiolabelled precursor of Tom40 at 25 °C for the indicated time periods. After lysis with digitonin, protein complexes were analysed by BN-PAGE and autoradiography. SAMsubstrate, Tom40 precursor bound to SAM complex; Int-II, Tom40 released from SAM; TOM, mature, assembled TOM complex6,20. Data are representative of two independent experiments. h, After heat treatment at 37 °C for 15 min, isolated wild-type and sam35-15 mitochondria were lysed with digitonin and incubated with glutathione–Sepharose beads that had been loaded with GST–β-signal fusion proteins, (GST)–β-signalPor1 (WT) or GST–β-signalPor1F281Q (FQ)6. The β-signal (β-strand 19) was released by thrombin protease and eluates were analysed by SDS–PAGE and immunodetection. Data are representative of three independent experiments.

Extended Data Fig. 7 The POTRA domain and overall structure of the SAMMdm10 complex.

a, Cartoon representation of the SAMMdm10 complex superimposed upon cryo-EM density maps contoured at 7.0σ above average (left) and 3.0σ above average (right). b, Cryo-EM density maps superimposed upon the atomic model of different parts of the POTRA domain, showing the fits of the density map and model. AA, amino acid. c, Modelled structure of the POTRA domain of the SAMMdm10 complex in nanodiscs. The POTRA domain was generated from the structure of BamA (PDB accession number 3Q6B; https://doi.org/10.2210/pdb3Q6B/pdb), and was modelled using molecular dynamics flexible fitting. The POTRA domain interacts with the loops connecting β-strands 2–3, 4–5 and 6–7 of the Sam50 barrel. Thus, the ‘POTRA domain density’ was visible only when lowering the threshold from 7.0σ to 3.0σ. Although the side chains of this density were not well resolved, a model with the Sam50 POTRA domain could be built by using comparative modelling and molecular dynamics flexible fitting. d, The N-terminal segment of Mdm10 penetrates into the β-barrel pore of Mdm10 and interacts with the luminal surface of the barrel (Fig. 3c and Extended Data Fig. 7d), resulting in a similar topological arrangement of the N terminus to that observed for the mature 19-stranded β-barrel proteins Tom40 and porin/VDAC16,17,69,70,71,72. Shown are interactions between the N-terminal segment (α-helices 1 and 2) and the inner wall of the Mdm10 β-barrel. e, f, Comparison of the determined Mdm10 structure (e; left) and that proposed previously by homology modelling28 (e; right). Notably, the β-strand arrangement of the determined Mdm10 structure deviates from that predicted by homology modelling28 (e) and from the fit of the models into the density maps for the critical parts, β-strands 3 and 14 (f). g, h, Hydrophobic interactions involving phospholipid-like molecules are shown by the cryo-EM densities in purple between Sam50a and Mdm10 β-barrel surfaces.

Extended Data Fig. 8 Modelling of Tom40 and Por1 onto the position of Mdm10 in the SAMMdm10 complex.

a, Contacts of the conserved residues between Mdm10 and Sam50a in the SAMMdm10 complex. Conserved residues of Mdm10 and Sam50a are represented by stick and space-filling forms, respectively. Residues in parentheses are the most frequently observed residues at each conserved position. Direct contacts between Mdm10 and Sam50a include interactions between Mdm10 residues with a bulky hydrophobic side chain and complementary Sam50a residues with a less bulky or hydrophobic side chain (left), and interactions between hydrophilic residues (right). b, Possible penetration of α-helices 6–8 of Sam37 into Sam50b, Mdm10, Tom40 and Por1. Loop 6 of Sam50b is shown in purple. The N and C termini of Tom40 and the N termini of Mdm10 and Por1 are shown in light colours. For Sam50b, penetration of α-helices 6–8 of Sam37 into the β-barrel pore would be prevented by the position of loop 6 of Sam50b. c, Por1 and VDAC structures70,71,72 are fitted onto the SAMMdm10 structure. There is no serious crush between Por1 or VDAC proteins and the SAM subunits (left), in particular between Sam37 and the strand 18–19 loop of Por1 or VDAC (red dotted oval on right), that could hamper complex formation. d, Conserved residues from the interaction interface between Mdm10 and Sam50. These conserved residues are shown on the Tom40 (PDB accession number 6JNF; https://doi.org/10.2210/pdb6JNF/pdb)16 and Por1 (structural model) structures, which were fitted to the Mdm10 structure in the SAMMdm10 complex. The residue labels are in the same colour when they are located at similar positions to the Mdm10 conserved residues. e, Structural model showing the interactions of Tom40 (with small Tom subunits; derived from the structure of the TOM complex)16 with Sam50a. Although the transmembrane segments of the small Tom subunits do not interfere with Tom40–Sam50a interactions, the position of the C-terminal short helix of modelled Tom7 is found at the interaction interface between Tom40 and Sam50a (red broken oval on right). Binding of the C terminus of Tom7 to Tom40 may thus be coupled to the release of Tom40 from the SAM complex27,28.

Extended Data Fig. 9 Analysis of SAMsubstrate complexes.

a, b, Chemical amounts of Tom40His were imported into wild-type mitochondria. Mitochondria were lysed with digitonin and subjected to affinity purification with Ni-NTA. Proteins were analysed by SDS–PAGE and immunodetection. Load 2%; eluate 100%; asterisk, nonspecific band; the top panel in b was decorated with anti-Mdm10 and anti-His antibodies. Data are representative of two independent experiments. c, Cells were treated with cycloheximide (CHX) as indicated. Isolated Sam50Cfree (CF) and Sam50C481 mitochondria were incubated with the chemical crosslinking reagent BMOE for 30 min on ice. After lysis, proteins were analysed by SDS–PAGE, western blotting and immunodetection with the indicated antibodies. Arrowhead, Cys-specific crosslinking product of Sam50; asterisk, nonspecific band. Data are representative of three independent experiments. d, Isolated wild-type mitochondria (with protein amount shown in μg) were analysed in parallel with the isolated His6–Por1β2–19–SAMsubstrate complex as in Fig. 4f. Data are representative of three independent experiments. e, The isolated His6–Por1β13–19–SAMsubstrate complex was analysed as in Fig. 4f and Extended Data Fig. 9d. Data are representative of three (for Sam37 and Mdm10) or four (for Sam35, Sam50, Tom22 and Fis1) independent experiments. f, Radiolabelled Por1 constructs were imported into wild-type mitochondria and analysed by BN-PAGE and autoradiography. POR, mature assembled porin complexes; SAMsubstrate, porin precursors arrested at SAM. Data are representative of three independent experiments. g, [35S]Por1β2–19 precursor proteins were imported into wild-type and Sam35His or ProtA–Sam37 mitochondria for 30 min, lysed with digitonin and incubated in the presence or absence of antibodies for 30 min. Lysed mitochondria were subjected to BN-PAGE and autoradiography. Data are representative of two independent experiments. h, [35S]Tom40 constructs were analysed as in f. SAM-Ia and Ib, SAMTom40 precursor intermediates; Int-II, Tom40 assembly intermediate II; TOM, mature, assembled Tom40. Data are representative of three independent experiments. i, Radiolabelled Tom40 was imported into isolated wild-type mitochondria for 3 min (pulse). After reisolation of mitochondria, imported Tom40 was chased for the indicated time periods. Mitochondria were lysed with digitonin, and protein complexes were analysed by BN-PAGE and autoradiography. Data are representative of two independent experiments. The full-length Tom40 precursor was present in the SAM complex for several minutes during the in organello assembly assay before its release and assembly into the TOM complex4,5,6,11,12,13,20,22,23,68,73.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics

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Takeda, H., Tsutsumi, A., Nishizawa, T. et al. Mitochondrial sorting and assembly machinery operates by β-barrel switching. Nature 590, 163–169 (2021). https://doi.org/10.1038/s41586-020-03113-7

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