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Cryo-EM structure of the mitochondrial protein-import channel TOM complex at near-atomic resolution

Nature Structural & Molecular Biology volume 26, pages 1158–1166 (2019)Cite this article

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Abstract

Nearly all mitochondrial proteins are encoded by the nuclear genome and imported into mitochondria after synthesis on cytosolic ribosomes. These precursor proteins are translocated into mitochondria by the TOM complex, a protein-conducting channel in the mitochondrial outer membrane. We have determined high-resolution cryo-EM structures of the core TOM complex from Saccharomyces cerevisiae in dimeric and tetrameric forms. Dimeric TOM consists of two copies each of five proteins arranged in two-fold symmetry: pore-forming β-barrel protein Tom40 and four auxiliary α-helical transmembrane proteins. The pore of each Tom40 has an overall negatively charged inner surface attributed to multiple functionally important acidic patches. The tetrameric complex is essentially a dimer of dimeric TOM, which may be capable of forming higher-order oligomers. Our study reveals the detailed molecular organization of the TOM complex and provides new insights about the mechanism of protein translocation into mitochondria.

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Fig. 1: Structure of the dimeric core TOM complex from S. cerevisiae.
Fig. 2: Intersubunit contacts between Tom40 and α-helical Tom subunits.
Fig. 3: Pore architecture of Tom40.
Fig. 4: Analysis of oligomeric states of the TOM complex.
Fig. 5: Cryo-EM structure of the tetrameric TOM complex.
Fig. 6: Model for presequence engagement with the TOM complex.

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Data availability

The cryo-EM density maps and atomic model are available through EM DataBank (EMD-20728, EMD-20729) and Protein Data Bank (PDB 6UCU, PDB 6UCV), respectively. Source data for Figs. 4d,e and 5d,e are available with the paper online.

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Acknowledgements

We thank D. Toso for help with electron microscope operation and J. Thorner for yeast strains and antibodies. We thank J. Thorner, J. Hurley, S. Brohawn, and S. Itskanov for critical reading of the manuscript. This work was funded by UC Berkeley (E.P. and J. T.), Vallee Scholars Program (E.P.), and NSF Graduate Research Fellowship Program (K.T.; DGE-1752814).

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Authors and Affiliations

  1. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA

    Kyle Tucker & Eunyong Park

  2. California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA, USA

    Eunyong Park

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  1. Kyle Tucker
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Contributions

E.P. conceived the project. K.T. and E.P. performed experiments. E.P. built the atomic models. K.T. and E.P. interpreted results and wrote the manuscript. E.P. supervised the project.

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Correspondence to Eunyong Park.

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Peer review information Katarzyna Marcinkiewicz was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Single-particle cryo-EM analysis of the dimeric core TOM complex.

a, Summary of single-particle image analysis procedure. b, A representative motion-corrected micrograph. Scale bar, 20 nm. Right panels show magnified images of selected particles outlined with white squares. The particle image size is 209 Å (width) by 209 Å (height). c, Representative class averages from 2D classification by RELION3. The box dimensions are 297 Å (width) by 297 Å (height). Classes in red boxes are likely empty micelles and thus excluded in subsequent analysis. d, Heat map showing particle orientation distribution (produced in the final 3D reconstruction by cryoSPARC2). e, Fourier shell correlation (FSC) of two independently refined half maps. Blue line, corrected masked FSC. Solid black line, unmasked FSC.

Extended Data Fig. 2 Cryo-EM map and atomic model quality of the dimeric TOM complex.

a, Local resolution represented by a heat map on the density contour (unsharpened, summed map). b, FSC between the EM map and the atomic model. Blue curve, FSCwork (FSC between half map 1 and a model refined against half map 1). Red curve, FSCfree (FSC between half map 2 and the model refined against half map 1). Black curve, FSCfull (FSC between the combined map and the final atomic model refined against the combined map. All refinements were performed by Phenix with the same weight. c, Examples of the density map and the atomic model for indicated segments. Numbers in the brackets indicate ranges of amino acid residues shown. d, Structural comparison of Tom40 and VDAC. Structures of Tom40 (this study; green) and murine VDAC (PDB 3EMN; magenta) are superimposed. Left, view from cytosol. Right, side view. e, Density features (green) for the C-terminal tails of Tom40 are shown in a 5-Å low-pass-filtered map. Shown is vertical cross-section along the Tom40 pores. A weak connection (not shown) between the density in green and α3 of Tom40 is indicated by a dashed line.

Extended Data Fig. 3 Surface complementarity of Tom subunits at interfaces and purification of the TOM complex with a K90A H102A mutation.

a, b, Overview of the dimeric TOM complex in solvent-accessible surface representation. Shown are views from the cytosol (a) and along the membrane plane (b). The color scheme is the same as in Fig. 1. The regions marked by a dashed line are magnified in c (with a 180° rotation) and d, respectively. c, Interface between the two Tom40 subunits. Left, a view from IMS (showing all subunits). Middle, as in the left panel but showing only Tom40 and DDM detergent molecules. Right, as in the middle panel but showing a side view. d,e, Side views showing the Tom40 and Tom22 interfaces within the same asymmetric unit (d) and between the two asymmetric units (e). f–h, Side view showing interfaces between Tom40 and other small Tom subunits. The viewing angles are the same as in Fig. 2b–d, respectively. i, Superose 6 SEC profile of the mutant TOM complex with K90A H102A Tom40 (solid blue) purified as the wild-type complex (dashed gray line; also see Fig. 1a).

Extended Data Fig. 4 Acidic and hydrophobic patches on the Tom40 pore surface.

a, Overview (side view) of the dimeric TOM complex (gray ribbons) and the Tom40 pore cavity (surface representation; shown for only one Tom40 subunit). b–d, Surface electrostatics is shown as a heat map overlaid on the pore cavity shown in surface representation. Side chains of acidic amino acids are shown in stick representation (AP1, AP2, and AP3 are in yellow, green, and pale blue, respectively). In c, only AP2 side chains are shown for clarity. e–g, As in b–d, but side chains of hydrophobic patches are shown in stick representation (HP1, HP2, are HP3 are in olive, green, and magenta, respectively). Note that some hydrophobic side chains in HP1 (labeled in gray; F81, F327, and L76) are only partially exposed, as they are involved in interactions between the α2 segment and the β sheets. h, As in Fig. 3j, but with mutants of AP3, HP2, and HP3. AP3mut1= E268N E308N; AP3mut2= E69N D73N; HP2mut1= L183S L216S; HP2mut2= V196N V198S; HP3mut1= L119S A121N F126N; HP3mut2= M94N A97S. Dox, doxycycline. i, Expression of Tom40 pore mutants (contains a C-terminal Strep-tag) was examined by SDS-PAGE and immunoblotting analyses of whole-cell lysates. PGK1, loading controls. Source data for i are available with the paper online. The experiments in h and i were repeated at least twice with similar results.

Source Data

Extended Data Fig. 5 Homology modeled pore architecture of N. crassa Tom40.

As in Fig. 3 a–d, but with N. crassa TOM complex. An N. crassa homology model was generated by SWISS-MODEL using the S. cerevisiae structure as a template, and electrostatic potential was calculated by Adaptive Poisson-Boltzmann Solver (APBS). The dashed yellow line indicates AP2. Note that unlike the S. cerevisiae TOM complex AP3 is not prominent in N. crassa.

Extended Data Fig. 6 Effects of detergent on the oligomeric state of the TOM complex.

a, Schematic diagram of the TOM complex purification procedure. Different detergent conditions (indicated by blue texts) were tested (specific conditions in b–e). b–e, Detailed SEC profiles of the purified TOM complex purified under different detergent conditions. “D” indicates the dimer peak, and “T” indicates the tetramer peak. Positions of the void peak (void) and peaks of molecular weight standards are indicated by arrowheads. TG, thyroglobulin (670 kDa); F, ferritin (440 kDa); ald, aldolase (156 kDa). Note that b–d is the same as in Fig. 4a–c, and b is the same experiment shown in Fig. 1a. f, SDS-PAGE analysis of peak fractions from the SEC purification shown in c. The peak positions are marked with “T” and “D”. The SDS gel was stained by Coomassie. g, Crude lysates prepared from cells overexpressing the TOM complex were solubilized with indicated detergent and subjected to BN-PAGE, followed by immunoblotting using an anti-Strep-tag antibody (detecting Tom40-Strep). A gradual decrease of mobility of the TOM complex accompanied by lowered detergent concentrations is likely due to an increased detergent micelle size. Source data for g are available with the paper online. The experiment in g was repeated twice with similar results.

Source Data

Extended Data Fig. 7 Cryo-EM analysis of the tetrameric complex.

a, Summary of single-particle image analysis procedure. b, A representative micrograph. Scale bar, 20 nm. The dimensions of magnified images are 414 Å (width) by 414 Å (height). c, Examples of selected 2D class averages. The box dimensions are 460 Å (width) by 460 Å (height). d, Particle orientation distribution. e, Fourier shell correlation (FSC). Blue line, corrected masked FSC. Solid black line, unmasked FSC. f, Local resolution map. g, Example images of particles larger than the tetramer. The leftmost image shows a 2D projection (side view with the longest width) of the 3D reconstruction of the tetrameric TOM complex. The other images show examples of large particles on micrographs. Estimated oligomeric states are indicated. Scale bar, 100 Å.

Extended Data Fig. 8 Dimer-dimer interface in the tetrameric TOM complex.

a, Overview (cytosolic view) of the tetrameric TOM complex. The 4.1-Å-resolution 3D reconstruction was represented with a composite map showing two different contour levels to show the protein features (colored contour; low-pass-filtered at 4.1 Å) and the detergent micelle (semitransparent gray contour; low-pass-filtered according to local resolution values). Organization of monomeric units are schematized in the upper right corner. Areas marked by dashed rectangles are shown in b and c (after rotating for a side view) with arrows and eye symbols indicating the viewing directions. b,c, Side views showing the dimer-dimer contacts between units B and C. Note that the tetramer is not symmetric and that there is a sizeable gap between Tom5B and Tom22C (c) in contrast to Tom5C and Tom22B (b). d, As in a, but showing a side view. Dashed lines indicate cross-sectional planes for cutaway views shown in e–g. e–g, Cutaway views (views from cytosol) at different positions along the membrane axis. In e and f, major interactions mediating the tetramerization are indicated by dashed ovals. Note that in g, there is a gap along the interface (also see h and i). h, As in a and d, but showing a view from IMS. i, Solvent-accessible surface of the tetrameric TOM complex. The dashed line indicates the interfacial gap. The two dimers (A–B and C–D) are in blue and red, respectively.

Extended Data Fig. 9 Biochemical validation of higher oligomeric TOM complexes.

a, Left, overview (angled cytosolic view) of the tetrameric TOM complex. Monomeric units B and C are shown in color, and A and D are in gray. The region in the black dashed box is magnified and shown in the right panel. Right, the cryo-EM density map (semitransparent gray) and the atomic model (in color) are shown for the B–C interface. The blue dashed arrows indicate the directions of the unmodeled N-terminal segments (residues 1–24) of the Tom6C and Tom6B subunits. The black dotted oval indicates the hydrophobic patch HP2. The green dashed lines indicate the unmodeled loop (L14-15; residues 277–294) between β14 and β15 of Tom40. b, Mitochondria were treated with BM-PEG2 and analyzed by SDS-PAGE and immunoblotting (IB). Tom40 contained no or an indicated single cysteine. c, Mitochondria were purified from cells expressing Tom40Strep (M287C) from the chromosomal locus and Tom40His (M287C) from a CEN plasmid. After treating with BM-PEG2, mitochondria were solubilized with octyl glucoside and subjected to immunoprecipitation (IP) using anti-Strep-tag antibodies (mock: IP without anti-Strep-tag antibodies). d, Mitochondria with Tom40Strep (M287C) expressed from the endogenous promoter were solubilized in 0.5% LMNG and 0.1% CHS and then injected to Superose 6 column. Fractions were treated with BM-PEG2 before SDS-PAGE and immunoblotting. e, As in Fig. 4e, but with mitochondria isolated from the tom6Δ mutant background. “T” and “D” indicate the peak positions of tetramers and dimers, respectively. Yeast were grown in YPEG (b and e) or YPD (c and d). Source data for panels b–e are available with the paper online. The experiments in b–e were repeated at least twice with similar results.

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Tucker, K., Park, E. Cryo-EM structure of the mitochondrial protein-import channel TOM complex at near-atomic resolution. Nat Struct Mol Biol 26, 1158–1166 (2019). https://doi.org/10.1038/s41594-019-0339-2

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