Abstract

The exocyst is an evolutionarily conserved octameric protein complex that mediates the tethering of post-Golgi secretory vesicles to the plasma membrane during exocytosis and is implicated in many cellular processes such as cell polarization, cytokinesis, ciliogenesis and tumor invasion. Using cryo-EM and chemical cross-linking MS (CXMS), we solved the structure of the Saccharomyces cerevisiae exocyst complex at an average resolution of 4.4 Å. Our model revealed the architecture of the exocyst and led to the identification of the helical bundles that mediate the assembly of the complex at its core. Sequence analysis suggests that these regions are evolutionarily conserved across eukaryotic systems. Additional cell biological data suggest a mechanism for exocyst assembly that leads to vesicle tethering at the plasma membrane.

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Acknowledgements

We are grateful to J. Lei, Y. Xu, Z. Yan, Q. Zhou, J. Wang and T. Yang for their help with cryo-EM experiments, structure determination and model building. We thank the Beijing National Protein Science Facility at Tsinghua University and Electron Microscopy Resource Laboratory at the University of Pennsylvania for support. This work is supported by a National Institute of Health grant (R01 GM111128) to W.G. and grants from the National Science Foundation of China (Grant 31530018), Beijing Municipal Science & Technology Commission (Grant Z161100000116034) and National Key Research and Development Program of MOST (Grant 2016YFA0501100) to H.-W.W.

Author information

Author notes

    • Jun-Jie Liu

    Present address: Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

  1. Kunrong Mei and Yan Li contributed equally to this work.

Affiliations

  1. Department of Biology, University of Pennsylvania, Philadelphia, PA, USA

    • Kunrong Mei
    • , Shaoxiao Wang
    • , Guangzuo Luo
    • , Peng Yue
    •  & Wei Guo
  2. Ministry of Education Key Laboratory of Protein Sciences, Tsinghua University, Beijing, China

    • Yan Li
    • , Jia Wang
    • , Jun-Jie Liu
    • , Xinquan Wang
    •  & Hong-Wei Wang
  3. Beijing Advanced Innovation Center for Structural Biology, Tsinghua University, Beijing, China

    • Yan Li
    • , Jia Wang
    • , Jun-Jie Liu
    • , Xinquan Wang
    •  & Hong-Wei Wang
  4. School of Life Sciences, Tsinghua University, Beijing, China

    • Yan Li
    • , Jia Wang
    • , Jun-Jie Liu
    • , Xinquan Wang
    •  & Hong-Wei Wang
  5. Tsinghua-Peking Joint Center for Life Sciences, Beijing, China

    • Yan Li
    • , Jia Wang
    •  & Hong-Wei Wang
  6. National Institute of Biological Sciences, Beijing, China

    • Guangcan Shao
    • , Yuehe Ding
    •  & Meng-Qiu Dong

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Contributions

W.G. initiated the project. W.G. and H.-W.W. designed and supervised the experiments. K.M., G.L., P.Y., and Y.L. purified the exocyst complex. Y.L. and K.M. collected the EM data. Y.L., H.-W.W., J.W., K.M. and J.-J.L. analyzed the EM data and generated the EM map. G.S., Y.D. and M.-Q.D. performed the CXMS experiments. K.M., G.S., Y.L., M.-Q.D., W.G. and H.-W.W. analyzed the CXMS data. K.M., Y.L., H.-W.W. and W.G. built the atomic model. K.M., H.-W.W., W.G. and X.W. analyzed the structure. S.W. performed the microscopy of Sec3 mutations. S.W. and K.M. performed the secretion assays. K.M., S.W. and W.G. analyzed the cell biology data. W.G., K.M. and Y.L. wrote the draft. W.G., K.M., H.-W.W. and X.W. edited the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Hong-Wei Wang or Wei Guo.

Integrated supplementary information

  1. Supplementary Figure 1 Flow chart of data processing of the cryo-EM dataset.

    Refer to the MATERIALS AND METHODS for details. The 3-D map in the red circle indicates a class with Sec3 in different conformation.

  2. Supplementary Figure 2 The cryo-EM data processing results.

    a. Cryo-EM 2-D class averages of the exocyst complexes purified from different strains with various subunit tagging. The red arrowheads indicate the density of C-terminally fused GFP in the corresponding complexes. CBD, calmodulin-binding domain. b. The full gallery of reference-free 2-D class averages that were included in the final 3-D reconstitution of the exocyst complex. The scale bar represents 20 nm. c. The angular distribution for the final 3-D reconstitution. The length of each cylinder is proportional to the number of particles for that view. d. The gold-standard FSC curve for the map refined with a mask around the body and distortion magnification correction. e. The Resmap of the 3-D reconstruction showing local resolutions.

  3. Supplementary Figure 3 Flow chart and summary of the model building.

    Regions built in each step are shown above the arrows, and modeling strategy is shown below the arrows. Back view maps are shown in the upper panel. Front view maps are shown in the lower panel. Also see Supplementary Video 1 for step-by-step exocyst model building and Supplementary Video 2 for CXMS analysis of the exocyst complex.

  4. Supplementary Figure 4 Rigid docking and model building of Sec6, Sec10, Sec15, Exo70 and Exo84.

    a-c. Rigid docking of Exo84 (a.a.525-753), Exo70 (a.a.67-623) and Sec6 (a.a.411-805), respectively. Exo70 (a.a. 67-623) was split into two domains for separate docking. Blue, Exo70 (a.a. 67-344); yellow, Exo70 (a.a. 345-623). The spheres in c indicate the Cα atoms of labeled lysine residues involved in the cross-linking. Distances between Cα-Cα of the crosslinked residues are shown above the dashed lines. d. Rigid docking of Sec10 based on Zebrafish Sec10(a.a.195-708) (PDB code: 5H11), Minimal adjustment was applied to fit the model into the map (Purple, original docking; cyan, docking with adjustment). e. Docking result of Exo84 PH domain based on rat Exo84(a.a.171-283) (PDB code: 1ZC4). f. Docking result of Sec15 C-terminal domain based on Drosophila Sec15(a.a.382-699) (PDB code: 2A2F). The map is set at high (left) and low threshold (right), respectively. g-k. Final models of Sec6, Sec10, Sec15, Exo70 and Exo84.

  5. Supplementary Figure 5 De novo model building of Sec5, Sec8 and Sec3.

    a-c. Model building of Sec5, Sec8 and Sec3. Close-up views show the cross-linking pairs that guided model building. Residue numbers are assigned based on secondary structure prediction. The spheres in each model indicate the Cα atoms of labeled residues in the cross-linking. Distances between Cα-Cα of the crosslinks are shown above the dashed lines (golden, Sec3; green, Sec5; purple, Sec6; forest green, Sec8; cyan, Sec10; blue, Exo70; pink, Exo84).

  6. Supplementary Figure 6 Representative EM density maps for the yeast exocyst complex.

    Several fragments of Sec5 (green), Sec15 (red) and Exo84 (pink) are shown, with the starting and ending residues labeled. Clear helical pitches can be seen in each of these fragments. While Sec5 and Sec15 are consisted of poly-alanine to trace the main chain, Exo84 (a.a. 525- 753) docked with crystal structure (PDB code: 2D2S) retains its side chains. Partial Exo84 side chains are displayed to show the consistency with the protuberances of the map.

  7. Supplementary Figure 7 Secondary structure prediction of the exocyst subunits.

    The diagram was generated with results from Predictprotein (brown, helix; blue, beta-strand). Amino acid numbers were shown in the scale bar above. The CorEx motifs of the exocyst subunits are indicated by black lines. The long linker region between the PH domain and the CorEx motif of Sec3 is indicated by the green line.

  8. Figure Supplementary 8 Docking of Sec3 PH domain suggests the plasma membrane-binding surface of the exocyst complex.

    The PH domain of Sec3 was docked to the rear bottom of the back layer based on its cross-linking with Sec15 (see text). The interface between the exocyst complex and the plasma membrane, as determined by the interactions of PI(4,5)P2 with Exo70 C-terminus and Sec3 PH domain (Yamashita, M. et al., Nat. Struct. Mol. Biol. 17, 180–186, 2010; He, B. et al., EMBO J. 26, 4053–4065, 2007), is modeled to the rear face of the back layer of the exocyst complex. The dash line connecting the PH domain and the built Sec3 model indicates the linker between the two regions. Golden, Sec3; blue, Exo70; red, Sec15. The red spheres mimic the phosphates of PI(4,5)P2.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–8, Supplementary Tables 1 and 2 and Supplementary Note 1

  2. Life Sciences Reporting Summary

  3. Supplementary Dataset 1

    Results of mass spectrometric analysis of DSS/BS3 cross-linked intermolecular residues in the exocyst complex

  4. Supplementary Dataset 2

    Results of mass spectrometric analysis of DSS/BS3 cross-linked intramolecular residues in the exocyst complex

  5. Supplementary Dataset 3

    Results of mass spectrometric analysis of EDC cross-linked residues in the exocyst complex

  6. Supplementary Dataset 4

    Raw SDS-PAGE and western blot images

  7. Supplementary Video 1

    Model building of the exocyst complex. Step-by-step model building process is shown. First, available crystal structures from yeast were docked into the map. Then, structures predicted from homology structures were docked and mildly adjusted to fit the map. Next, de novo model extension was performed based on the map connectivity and secondary structure prediction. After that, the density for Sec5 and Sec8 was assigned with the guidance of the CXMS information and de novo model building was performed based on map connectivity and secondary structure prediction. Finally, the remaining density was assigned to Sec3 and its model was manually built as did with other subunits.

  8. Supplementary Video 2

    CXMS analysis of the exocyst complex. All confident (E value<1E–4 and counts>2) intermolecular and intramolecular residue pairs cross-linked by BS3, DSS and EDC are mapped onto the exocyst model. Crosslinks with Cα–Cα distance> 35 Å are displayed as outliers. In total, 280 crosslinks are shown, of which 11 are outliers.

  9. Supplementary Video 3

    The assembly of the exocyst complex. The hierarchical assembly of the exocyst complex is displayed. First, Sec3–Sec5, Sec6–Sec8, Sec10–Sec15 and Exo70–Exo84 formed heterodimer through their CorEx motifs. Then the four-helical bundle formation with the CorEx motifs of Sec3–Sec5–Sec6–Sec8 and Sec10–Sec15–Exo70–Exo84 results in the subcomplex 1 and subcomplex II. Finally, the holo-exocyst complex was assembled through the interactions between subcomplex I and subcomplex II.

Source data

  1. Source Data, Figure 6c

    Source data for Figure 6c

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https://doi.org/10.1038/s41594-017-0016-2