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Architecture of chloroplast TOC–TIC translocon supercomplex

Abstract

Chloroplasts rely on the translocon complexes in the outer and inner envelope membranes (the TOC and TIC complexes, respectively) to import thousands of different nuclear-encoded proteins from the cytosol1,2,3,4. Although previous studies indicated that the TOC and TIC complexes may assemble into larger supercomplexes5,6,7, the overall architectures of the TOC–TIC supercomplexes and the mechanism of preprotein translocation are unclear. Here we report the cryo-electron microscopy structure of the TOC–TIC supercomplex from Chlamydomonas reinhardtii. The major subunits of the TOC complex (Toc75, Toc90 and Toc34) and TIC complex (Tic214, Tic20, Tic100 and Tic56), three chloroplast translocon-associated proteins (Ctap3, Ctap4 and Ctap5) and three newly identified small inner-membrane proteins (Simp1–3) have been located in the supercomplex. As the largest protein, Tic214 traverses the inner membrane, the intermembrane space and the outer membrane, connecting the TOC complex with the TIC proteins. An inositol hexaphosphate molecule is located at the Tic214–Toc90 interface and stabilizes their assembly. Four lipid molecules are located within or above an inner-membrane funnel formed by Tic214, Tic20, Simp1 and Ctap5. Multiple potential pathways found in the TOC–TIC supercomplex may support translocation of different substrate preproteins into chloroplasts.

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Fig. 1: The overall architecture of the TOC–TIC supercomplex.
Fig. 2: Structure of the Toc75–Toc90–Toc34 complex associated with Tic214.
Fig. 3: Structural role and components of the ISC.
Fig. 4: Arrangement of protein subunits and lipid molecules in the inner-membrane complex.
Fig. 5: The intrinsic pores found in the TOC and TIC complexes.

Data availability

The cryo-EM maps of the C. reinhardtii TOC–TIC supercomplex and the TOC complex, and their corresponding atomic coordinates, have been deposited in the Electron Microscopy Data Bank and the PDB under accession codes EMD-33528 and 7XZI, and EMD-33529 and 7XZJ, respectively. The other PDB files used for analysis and comparison in this work were downloaded from the RCSB PDB website (http://www.rcsb.org/), including the structures of A. thaliana Toc75 POTRA domains (5UAY), the NMR structure of the C. reinhardtii preFdx1 transit peptide (1FCT) and the crystal structure of RopP2 from Ralstonia solanacearum (5W3X). The full versions of all gel images are provided in Supplementary Data 1. All data analysed during this study are included in the Article and its Supplementary InformationSource data are provided with this paper.

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Acknowledgements

We thank X.-J. Huang, B.-L. Zhu, X.-J. Li, D.-Y. Fan, L.-L. Zhang and the other staff members at the Center for Biological Imaging (CBI), Core Facilities for Protein Science at the Institute of Biophysics, Chinese Academy of Science (IBP, CAS) for the support in cryo-EM data collection; Z.-S. Xie, X. Ding, L.-L. Niu and J.-F. Wang at the CAS Research Platform for Protein Sciences for support with MS data collection and analysis; X.-B. Liang, Y.-X. Liu and X.-Y. Liu for technical support with biochemistry, cell culture and sample preparation; J.-Z. Lou and Y. Z. for their help in molecular dynamics simulations; S. Ramundo for sharing the HA-tagged Tic214 strain of C. reinhardtii; and W. Yang and N. Wang for recovering and aliquoting the cells. This work was funded by the Chinese Academy of Sciences Project for Young Scientists in Basic Research (YSBR-015), the National Natural Science Foundation of China (31925024), the Strategic Priority Research Program of CAS (XDB37020101 to Z.L.) and the National Key R&D Programme of China (2017YFA0503702).

Author information

Authors and Affiliations

Authors

Contributions

H.L. expressed and purified the protein complex sample, prepared the sample for the cryo-EM study, carried out cryo-EM data collection and prepared the figures. H.L. and A.L. processed the cryo-EM data. Z.L. and H.L. built and refined the atomic model, analysed the structure. H.L. designed the MS study and prepared the sample for the MS analysis. A.L. performed MD simulations on the TIC complex. Z.L., H.L. and A.L. wrote the initial draft and all of the authors edited the manuscript. J.-D.R. was involved in designing the project. Z.L. conceived and coordinated the research project.

Corresponding author

Correspondence to Zhenfeng Liu.

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The authors declare no competing interests.

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Nature thanks Alexey Amunts 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 and characterization of the TOC-TIC supercomplex from C. reinhardtii.

a, A schematic diagram illustrating the overall flowchart of the protein purification and cryo-EM grid preparation protocol. b, The profiles of size exclusion chromatography loaded with the sample eluted from the Anti-DYKDDDDK beads. The peak 1 fractions marked in the cyan area were pooled, concentrated and used for cryo-EM grid preparation. The blue solid line and green solid lines indicate the absorbance measured with the UV-Vis detector at 280 nm or 488 nm (the values and units are labelled on the Y axes on left and right sides) respectively. The grey dashed line is the size exclusion chromatography of the Gel Filtration Cal Kit High Molecular Weight sample (28-4038-42, Cytiva) loaded as a reference. c and d, The SDS-PAGE (c) and blue-native PAGE (d) analyses on the peak 1 fraction from the size exclusion chromatography. Silver staining of the gel was applied for visualization of the proteins. The identities of the protein bands are labelled on the right side according to the identification results obtained through mass spectrometry on the in-gel tryptic digestion products. Note that one of the FtsH-like AAA proteins named Ctap1 is present in the sample (d) and might be loosely associated with the TIC complex, but became separated from the TOC-TIC supercomplex during the purification process. For gel source data, see Supplemental Data 1. The SDS-PAGE and blue-native PAGE experiments were independently repeated three times and twice respectively with similar results. e, A representative cryo-EM micrograph of the TOC-TIC supercomplex. Scale bar, 20 nm. A total of 9,939 independent images of similar quality were collected from one cryo-EM grid and processed further.

Source Data

Extended Data Fig. 2 Cryo-EM data processing of the TOC-TIC supercomplex.

a, Flowchart of the cryo-EM data processing and analysis of the TOC-TIC supercomplex. (Please refer to the ‘Cryo-EM data processing and refinement’ section in Methods for more technical details). The two datasets were collected at different spots of the same grid sample. b and d The gold-standard Fourier shell correlation (GSFSC) curves of the TOC-TIC supercomplex map (b) and the local map around the TOC complex. c, The posterior precision directional distribution plot of the particles of the final reconstruction generated by cryoSPARC. e, The scheme for improving the densities around the TOC complex by applying the particle subtraction and local refinement procedure. f, The local resolutions for different regions of the final overall reconstruction of the TOC-TIC supercomplex. The local resolutions were estimated by cryoSPARC and visualized in ChimeraX. g, The densities of the detergent micelles surrounding the transmembrane domains of the TOC complex, Ctap4 and the TIC complex. The protein subunits are coloured in the same way as in Fig. 1, while the detergent micelles are in transparent white.

Extended Data Fig. 3 Comparison of the POTRA domains of C. reinhardtii Toc75 with the corresponding ones from plant Toc75.

a and b, Superposition of the cryo-EM structure of CrToc75 POTRA domains with the crystal structure of Arabidopsis Toc75 POTRA domains (PDB: 5UAY). The superposition was generated by using Chimera X matchmaker. c, Alignment of the amino acid sequences of the POTRA domains of Toc75 from Chlamydomonas reinhardtii, Arabidopsis thaliana, Spinacea oleracea and Pisum sativum. The sequence alignment was carried out by using the CLC Sequence Viewer 8. The β strands and α helices are represented by the arrows and cylinders respectively. Note that AtToc75 has an α-helix (marked in purple dotted circle in a and the purple cylinder in c) after the first β sheet of POTRA 2, which is not found in CrToc75 and has a sequence largely different from the corresponding region in CrToc75.

Extended Data Fig. 4 Domain organization, secondary structure topology and cryo-EM densities of Toc75, Toc90 and Toc34.

a, Domain organization of Toc75 protein. b, Topology of the secondary structures in Toc75. c, Cryo-EM densities of various parts in Toc75. The protein is built as chain 7 in the structural model and labelled as 7. The pore-lining mβ1-mβ5 region is highlighted in blue. d, Domain organization of Toc90 protein. e, Topology of the secondary structures in Toc90. f, Cryo-EM densities of various parts in Toc90. Chain 9 in the structural model corresponds to Toc90 (labelled as 9). The pore-lining α3-mβ1 loop, mβ1-mβ4, mβ13-mβ14 regions are highlighted in magenta, orange and green. g, Domain organization of Toc34 protein. h, Topology of the secondary structures in Toc34. i, Cryo-EM densities of various parts in Toc34. Chain G in the structural model corresponds to Toc34 (labelled as G).

Extended Data Fig. 5 The cryo-EM densities in the cytoplasmic region above the TOC complex and the structure of Ctap4.

a, The cryo-EM map of TOC-TIC supercomplex filtered through the Gaussian filter. The contour level is at 0.011 (Chimera). Apparently, there are some weak cryo-EM densities above the TMDs of the Toc75-Toc90-Toc34 complex, potentially belonging to the G domains of Toc90 and Toc34. The densities inside the elliptical dashed ring may contain the putative G-domains of Toc90 and Toc34. The G domains are likely very mobile as the densities are much weaker than the transmembrane domains and the loops connecting them to the transmembrane domains are barely visible in the map. b, Fitting of the cytosolic domain densities with a heterodimeric model of the G domains from Toc34 and Toc90. The model was constructed by referring to the homodimeric structure of Toc34 G domain (PDB code: 3BB1). The map contour level is at 0.0125. Colour codes: red, G-domain of Toc34; blue, G-domain and the N-terminal domain (NTD) of Toc90. c, Domain organization of the Ctap4 protein. d, Topological arrangement of the secondary structures in Ctap4 as predicted by Alphafold2. e, Cryo-EM densities of Ctap4 (chain 4). f and g, Side view and top view of Ctap4 structure. HM1 and HM2, hydrophilic motifs 1&2.

Extended Data Fig. 6 Location and identification of an InsP6 molecule in the TOC-TIC supercomplex from C. reinhardtii.

a, Electrostatic surface representation of the TOC complex hosting an InsP6-binding site. The zoom-in view on the right side shows an InsP6 molecule accommodated in a positive charge-rich cavity. The electropositive surface of the cavity is complementary to the negative charges carried by the phosphate groups of InsP6. b, The cryo-EM density of the InsP6 molecule with the stick model superposed. The cryo-EM map is contoured at 0.25 V. c, Interactions of InsP6 with adjacent amino acid residues from Tic214 (green) and Toc90 (blue) in the TOC-TIC supercomplex. d and e, The 2D interaction map of InsP6 in the TOC-TIC supercomplex (d) or RsPopP2 (PDB:5W3X, e) calculated by using the MOE program. RsPopP2 is a bacterial effector protein produced by the plant pathogen Ralstonia solanacearum and hosts an InsP6-binding site similar to the one found in the TOC complex. The phosphate groups of InsP6 molecules are surrounded by positively-charged residues in both cases. f and g, Mass spectrometry analysis of the extract from the TOC-TIC supercomplex sample (f) or the standard sample of phytic acid (sodium salt, g).

Extended Data Fig. 7 The assembly and components of the intermembrane space complex in the TOC-TIC supercomplex.

a, The assembly interfaces between ISC and the TOC/TIC complex. b, Domain organization and cryo-EM density of the intermembrane space domain of Tic214. c, Domain organization and cryo-EM density of Tic100. d, Domain organization, density and cartoon model of Tic56. e, Domain organization, densities and cartoon models of Ctap3 and Ctap5. Ctap3 has an extended α-helical domain (Arg295-Ser472) at the carboxy-proximal region, intertwining with the bowl-like region of Tic214-Tic100 complex. It docks two short α-helices (α1 and α2) on the outer surface of the upper half of ISC, inserts a long α-helix (α3) and two short ones (α4 and α5) into the bottom of the bowl-like structure formed by Tic214 and Tic100. The last three α-helices (α7-9) of Ctap3 bind to the amino-terminal helix of Tic56 and the carboxy-terminal helix of Tic100. On the opposite side, Ctap5 attaches its carboxy-proximal α-helical domain (Glu215-Ala329) to a surface groove of the Tic100-Tic214 complex below the POTRA1 and POTRA2 domains of Toc75. It interacts closely with the BJD of Tic100 and Tic214 (Gln1621-Phe1645 and Tyr1917-Ile1949) and provides binding sites for the POTRA1 and POTRA2 domains of Toc75. Thereby, Ctap5 may help to stabilize the Tic214-Tic100 complex and serves as a bridge to connect Toc75 with the inner-membrane complex.

Extended Data Fig. 8 Topology and cryo-EM densities of the six proteins involved in forming the TIC complex.

a, Topological arrangement of secondary structures in Ctap5 (chain 5), Simp2 (chain U), Tic214 (chain A), Tic20 (chain B), Simp3 (chain D) and Simp1 (chain C) proteins. b-g, Cryo-EM densities of various parts in the six proteins. h-j, Cartoon models of Simp1, Simp2 and Simp3.

Extended Data Fig. 9 The lipid and detergent molecules associated with the TOC-TIC supercomplex.

a, The cryo-EM densities of four central lipid molecules located in or above the putative pore of the TIC complex. b, Interactions of the four lipid molecules with adjacent amino acid residues. Green, Tic214; Pink, Tic20; brown, Ctap5. c, LigPlot analyses showing the hydrogen bonds and hydrophobic interactions formed between the four lipid molecules and nearby amino acid residues. d, The cryo-EM densities of other peripheral lipid molecules associated with Tic214(A), Tic20(B), Simp3(D), Ctap5(5) and Toc75(7).

Extended Data Fig. 10 The surface presentations of the TOC complex, the intermembrane space domain connecting the TOC channel with the TIC channel, and the TIC complex.

a and b, The electrostatic potential surface of the TOC complex viewed from the cytoplasmic and intermembrane space sides, respectively. c, The electrostatic potential surface of the ISC connecting the TOC complex with the TIC complex. d, The presence of a surface groove connecting the channels of the TOC and TIC complexes. The local regions labelled by “C” and “Δ” are the cavity above the central funnel of the TIC complex and the triangular entrance formed by Ctap5, Simp2 and Tic214, respectively. e and f, The electrostatic potential surface of the TIC complex viewed from the intermembrane space and stromal sides, respectively.

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

Supplementary information

Supplementary Information

Supplemental Data 1: uncropped raw images of the SDS–PAGE and blue native PAGE gels. Supplementary Table 1: a summary of each individual protein subunit identified in the TOC–TIC supercomplex from C. reinhardtii. Supplementary References: references cited in Supplementary Table 3.

Reporting Summary

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Supplementary Table 2

A full list of proteins identified in the TOC–TIC supercomplex sample by MS.

Supplementary Table 3

A list of various protein components of the TOC and TIC complexes.

Supplementary Video 1

The 3D variability of the TOC–TIC supercomplex of C. reinhardtii.

Supplementary Video 2

Nine different motion components of the TOC–TIC supercomplex revealed by the multibody analysis.

Supplementary Video 3

Molecular dynamics simulation on the TIC complex embedded in a lipid bilayer.

Supplementary Video 4

Molecular dynamics simulations on the TIC complex with the transit peptide of pre-ferredoxin 1 docked on the surface.

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Liu, H., Li, A., Rochaix, JD. et al. Architecture of chloroplast TOC–TIC translocon supercomplex. Nature 615, 349–357 (2023). https://doi.org/10.1038/s41586-023-05744-y

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