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In situ architecture of the ER–mitochondria encounter structure

Nature volume 618, pages 188–192 (2023)Cite this article

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Abstract

The endoplasmic reticulum and mitochondria are main hubs of eukaryotic membrane biogenesis that rely on lipid exchange via membrane contact sites1,2,3, but the underpinning mechanisms remain poorly understood. In yeast, tethering and lipid transfer between the two organelles is mediated by the endoplasmic reticulum–mitochondria encounter structure (ERMES), a four-subunit complex of unresolved stoichiometry and architecture4,5,6. Here we determined the molecular organization of ERMES within Saccharomyces cerevisiae cells using integrative structural biology by combining quantitative live imaging, cryo-correlative microscopy, subtomogram averaging and molecular modelling. We found that ERMES assembles into approximately 25 discrete bridge-like complexes distributed irregularly across a contact site. Each bridge consists of three synaptotagmin-like mitochondrial lipid binding protein domains oriented in a zig-zag arrangement. Our molecular model of ERMES reveals a pathway for lipids. These findings resolve the in situ supramolecular architecture of a major inter-organelle lipid transfer machinery and provide a basis for the mechanistic understanding of lipid fluxes in eukaryotic cells.

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Fig. 1: The number of molecules of ERMES components per MCS.
Fig. 2: ERMES-mediated MCS consist of bridge structures connecting the two membranes.
Fig. 3: Supramolecular organization of ERMES within MCS.
Fig. 4: Integrative modelling of the ERMES complex.

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Data availability

Data generated in this study have been deposited at the Electron Microscopy Data Bank76 (www.ebi.ac.uk/emdb) under accessions EMD-15355, EMD-16871, EMD-16872 and EMD-16873. Raw cryo-ET data has been deposited at EMPIAR77 (https://www.ebi.ac.uk/empiar) under accession EMPIAR-11462. Live fluorescence microscopy data and integrative molecular models have been deposited at www.zenodo.org at https://doi.org/10.5281/zenodo.6784812, https://doi.org/10.5281/zenodo.7392153, https://doi.org/10.5281/zenodo.7753491 and https://doi.org/10.5281/zenodo.7736245. Source data are provided with this paper.

Code availability

The SpotQuant package20 used for quantification of fluorescence intensities is available at https://github.com/apicco/spotquant and https://github.com/apicco/spotquant/tree/b10. The STA package subTOM50 is available at https://github.com/DustinMorado/subTOM. Other code available is for conversion of Dynamo dipole model coordinates to a MOTL file https://github.com/mwozn/DYNAMO_dipoles_to_MOTL, calculation and classification of bridge lengths from dipole ER- and OMM anchor points https://github.com/mwozn/getDipoleLength_binByLength, nearest-neighbour analysis https://github.com/mwozn/MOTL_neighbourAnalysis, and fluorescent protein levels on mitochondria https://github.com/mwozn/totalFluorPerCell.

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Acknowledgements

The authors thank the MRC LMB facilities for light microscopy, electron microscopy and scientific computing for support with data collection and processing; M. Kaksonen for encouragement and helpful discussions; and the Microscopy Imaging Center (MIC) of the University of Bern for support. M.R.W. was supported by the Natural Sciences and Engineering Research Council (NSERC) of Canada (PGSD). This work was supported by the Medical Research Council, as part of United Kingdom Research and Innovation (also known as UK Research and Innovation) under awards MC_UP_1201/8 to W.K. and MC_UP_1201/10 to E.A.M. For the purpose of open access, the authors have applied a CC BY public copyright licence to any Author Accepted Manuscript version arising. Work in the group of W.K. was also supported by the NCCR TransCure, a National Center of Competence in Research of the Swiss National Science Foundation (SNSF) (185544) and the SNSF project funding (201158). Work in the group of S.V. was supported by the SNSF (194807) and by the European Research Council under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 803952). This work was supported by grants from the Swiss National Supercomputing Centre under project ID s1132.

Author information

Author notes

  1. Michael R. Wozny

    Present address: Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada

  2. Dustin R. Morado

    Present address: Max Planck Institute of Biochemistry, Martinsried, Germany

  3. Patrick C. Hoffmann

    Present address: Max Planck Institute of Biophysics, Frankfurt am Main, Germany

Authors and Affiliations

  1. MRC Laboratory of Molecular Biology, Cambridge, UK

    Michael R. Wozny, Dustin R. Morado, Patrick C. Hoffmann, Elizabeth A. Miller & Wanda Kukulski

  2. Department of Biology, University of Fribourg, Fribourg, Switzerland

    Andrea Di Luca, Pablo Campomanes & Stefano Vanni

  3. SciLifeLab, Solna, Sweden

    Dustin R. Morado

  4. Department of Biochemistry, University of Geneva, Geneva, Switzerland

    Andrea Picco

  5. Institute of Biochemistry and Molecular Medicine, University of Bern, Bern, Switzerland

    Rasha Khaddaj, Lazar Ivanović & Wanda Kukulski

  6. Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland

    Lazar Ivanović

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Contributions

M.R.W. generated yeast strains, collected data for quantification of numbers of molecules, prepared cellular lamellae and collected cryo-CLEM and cryo-ET data. A.D.L. and P.C. performed the computational integrative modelling, supervised by S.V. A.D.L., P.C. and S.V. analysed the integrative modelling data. D.R.M. and M.R.W. developed the STA alignment procedure. M.R.W. performed STA with help from D.R.M. A.P. and M.R.W. implemented the quantitative live-cell imaging pipeline and analysed the resulting data. M.R.W., W.K. and E.A.M. analysed cryo-ET data and conceived the experimental strategy of the project. R.K. and L.I. participated in yeast strain generation, collected and analysed fluorescence imaging data and assessed yeast mutants. P.C.H. participated in yeast strain generation, cryo-FIB milling and cryo-ET data acquisition. W.K. conceived and supervised the project. M.R.W. and W.K. wrote the paper, with direct help by E.A.M., A.D.L. and S.V., as well as input from all authors.

Corresponding authors

Correspondence to Stefano Vanni or Wanda Kukulski.

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Extended data figures and tables

Extended Data Fig. 1 Correlation between simultaneously labelled ERMES components.

a: Fluorescence live cell microscopy of cells expressing Mdm34-eGFP and Mdm12-mCherry. Individual channels and merge are shown. b: Fluorescence live cell microscopy of cells expressing Mdm34-eGFP and Mmm1-mCherry. Individual channels and merge are shown. c: The fluorescence intensity of Mdm34-eGFP and Mdm12-mCherry, labelled in the same cells, correlates in puncta. n = 128 puncta, Pearson’s correlation R = 0.566, p = 3.38*10−12 (two-sided). d: The fluorescence intensity in a.u. of Mdm34-EGFP and Mmm1-mCherry, labelled in the same cells, correlates in puncta. n = 134 puncta, Pearson’s correlation R = 0.529, p = 5.18*10−11 (two-sided). Scale bars are 3 µm.

Source Data

Extended Data Fig. 2 MCS imaged by cryo-ET at Mdm34-mNeonGreen signals.

a—f: Diversity of membrane morphologies of ER-mitochondria MCS imaged by cryo-ET at locations of Mdm34-mNeonGreen. Six representative examples from a data set of 51 tomograms are shown. Left panels are virtual slices through electron cryo-tomograms. ER-mitochondria MCS are indicated by white arrows. Right panels are segmentation models of the ER (blue), the OMM (yellow), the IMM (mustard) and peroxisomes (pink). The segmentation models are rotated relative to the virtual slices to better visualize the MCS. The example in e is the same as shown in Fig. 3f. g, h: ER-peroxisome contacts imaged by cryo-ET at locations of Mdm34-mNeonGreen. Approximately 15% of Mdm34-mNeonGreen puncta contained ER-peroxisome MCS (indicated by white arrows) rather than ER-mitochondria MCS. Peroxisomes are identified as spheroid single-membrane vesicles more than 100 nm in diameter with dense interior78,79. Scale bars are 100 nm. Note that due to the perspective view, scale bars in the panels showing segmentation models apply only to the front plane of the scenes.

Extended Data Fig. 3 Subtomogram averaging pipeline.

The strategy using sequential alignment steps is outlined in steps a—h. The data set consisted of manually picked coordinates of the ER and OMM anchor points of 1098 bridge structures from 51 electron cryo-tomograms at positions of Mdm34-mNeonGreen signals. The resolution of the final STA map is potentially affected by a minor fraction of bridge-like particles of different identity, which could contribute noise. Images in a are the same as shown in Fig. 2b and c, with modifications to the overlay. Scale bars in a are 100 nm (left image) and 20 nm (images in pink dashed boxes).

Extended Data Fig. 4 Resolution estimate of STA map.

a: Fourier shell correlation (FSC), calculated according to59. The final resolution corresponds to the value at FSC = 0.143. b: Left panel: Three virtual slices through the unmasked STA map. Right panel: Three virtual slices through the STA map masked for FSC calculation.

Extended Data Fig. 5 STA maps of short and long bridges.

a, b: STA map of short class (pink). c, d: STA map of long class (teal). The two classes were obtained by halving the data set of 1098 bridge structures according to bridge length and subjecting both classes separately to the procedure shown in Extended Data Fig. 3. In a and c the maps are shown at lower contour levels than in b and d. Scale bars are 5 nm. Note that due to the perspective view, scale bars apply only to the front plane of the scenes.

Extended Data Fig. 6 Orientation of the bridges relative to the membranes.

a—d: STA alignment strategy to determine the angle between the bridges and the OMM. e: This graph is the same as shown in Fig. 3d. Large point indicates median, vertical lines MAD. n = 1098 bridges from 51 tomograms. f—i: STA alignment strategy to determine the angle between the bridges and the ER. j: This graph is the same as shown in Fig. 3e. Large point indicates median, vertical lines MAD. n = 1098 bridges from 51 tomograms. k: Comparison of the STA maps obtained from different alignments. Grey: Full STA map obtained from the alignment strategy depicted in Extended Data Fig. 3. The STA map is the same as shown in Fig. 2f and g, displayed at different contour level. Green: STA map obtained from the alignment strategy used to determine the angle between bridges and OMM, as depicted in panels a-d. Red: STA map obtained from the alignment strategy used to determine the angle between bridges and ER, as depicted in panels f—i. Scale bars are 5 nm. Note that due to the perspective view, scale bars apply only to the front plane of the scenes.

Extended Data Fig. 7 The distribution of bridges within the MCS ultrastructure.

a—c: Segmentation models of MCS in three different tomograms, with STA maps of short (pink) and long (teal) classes placed back to the tomographic positions of individual bridges. OMM is in yellow, IMM in mustard, and ER in transparent light blue. d: Distribution of bridges of the long and short classes, across MCS. Each dot represents one MCS (n = 64), plotted according to the percentage of long (left y-axis) and short bridges (right y-axis) it contains. e: Distribution of bridges of the long and short classes, within MCS. The number of short (left bar) and long (right bar) bridges that have a nearest neighbour belonging to the long (light grey bar fraction) and short (dark grey bar fraction) class. f: Dot plot and half-violin plot of the surface area of the ER membrane serviced by one ERMES bridge, determined per MCS. Large point indicates median, vertical lines indicate MAD. One outlier is not shown but included in median and MAD determination. n = 63 MCS from 49 tomograms.

Source Data

Extended Data Fig. 8 Integrative modelling of the ERMES complex.

a: Differences between FD (in colour) and AF multimer (grey) predicted structures. While initial predictions of the complex structure using AF multimer27,65 and FD26 yield a nearly identical Mmm1-Mdm12 interface, the Mdm12-Mdm34 FD-interface shows a larger aperture (black arrowheads) which tends to close upon fitting to the cryo-ET STA map (black arrow). This difference between AF and FD highlights a lack of dynamic information as a current limitation of structural prediction. b—d: Quality estimation of the dimer predictions (Mmm1/Mdm12, Mdm12/Mdm34, Mdm34/Mdm10) using the FoldDock (FD) protocol. For each protein, the local pLDDT score is shown, together with the overall DockQ score of the dimer. For all protein-protein interfaces, the DockQ score is above the value generally considered to successfully predict heteromeric interfaces (DockQ > 0.23)26,65. e: Final conformations of the trimeric Mmm1-Mdm12-Mdm34 complex, obtained from the MDFF simulations including 4 POPE lipids after fitting with different scaling factors (g_scale, numerical values given below models). The scaling factor determines the weight of the experimental STA map on the total molecular potential. f: Assessment of MDFF-derived models obtained using different scaling factors, shown in panel e. ccc refers to the cross-correlation coefficient between map and model, voroMQAtotal and voroMQAinter refer to the global voroMQA score and the component including only inter-subunit contacts, respectively. Based on the gscore which combines the assessment parameters (see Methods), g_scale = 0.3 was considered best and is thus shown in other figures. g: Models obtained when the conformation of the Mmm1-Mdm12-Mdm34 complex was biased by MDFF into the short (left) and long (right) STA maps. The interaction between Mmm1 and Mdm12 (indicated by black arrows) appears to be diminished in the long conformation. In both cases, the model contained 4 lipids and the scaling factor was g_scale = 0.1. See also Supplementary Table 1.

Extended Data Fig. 9 Effect of bound lipids on the ERMES complex model.

a: Model of the Mmm1-Mdm12-Mdm34 complex obtained by MDFF when the starting complex contained 4 POPE lipid molecules (left) as compared to 12 POPE lipid molecules (right). Note that the MDFF-derived model with 4 POPE molecules is also shown in Fig. 4a,b and c as well as in Extended Data Fig. 8e (g_scale = 0.3), without visualization of the lipids. b: Distributions of detected cavity radii at the interfaces between subunits Mmm1-Mdm12 (magenta) and Mdm12-Mdm34 (green) in the presence of 12 lipid molecules. The radii were computed from frames taken over the last 100 ns of the MDFF simulation and are shown as probability density distributions. c: Structural comparison between the heterotrimeric complex obtained by MDFF with the STA map (blue), and the same complex after an additional unbiased MD simulation of 120 ns. The left model is with 4 lipids bound, the right model with 12 lipids bound. d: Root mean square deviations (RMSD) of individual subunits and of the heterotrimeric complex, measured over the additional 120 ns unbiased MD simulations of the MDFF-obtained heterotrimeric complex with either 12 or 4 lipids bound (red and orange, respectively). The end points (120 ns) of the red and orange plots correspond to red and orange conformations of the complex shown in d, respectively. The differences in the RMSD of Mmm1 with 12 vs. 4 lipids can be attributed to its N-terminal region, see also Supplementary Fig. 2.

Extended Data Fig. 10 Mdm10 mutation in the predicted Mdm10-Mdm34 interface.

a: MDFF-derived predicted interface between Mdm10 (blue) and Mdm34 (green). Residues in wild type Mdm10 that were mutated to Mdm10W238A/G240L/L274A/F275A to disrupt the interface are indicated in yellow. The mutated residues are not involved in known interactions within the SAM complex21,80. b: Spot growth assay on YPD, comparing wild type, Mdm10Y296A/F298A/Y301A (shown before to disrupt the interface between Mdm10 and Mdm3421) and Mdm10W238A/G240L/L274A/F275A. All three strains express Tom20-Cherry. c: Light microscopy of strains expressing wild type and mutant Mdm10. Tom20-mCherry is used as an indicator of mitochondrial morphology. Mdm10Y296A/F298A/Y301A and Mdm10W238A/G240L/L274A/F275A display a similar mitochondrial phenotype, indicative of ERMES complex disruption4,21. Scale bars are 3 µm. See also Supplementary Fig. 3.

Extended Data Table 1 Cryo-ET data collection and processing
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Supplementary information

Supplementary Information

This file contains Supplementary materials and methods, Supplementary Figs. 1–3 and Supplementary Table 1.

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Supplementary Video 1

The STA map calculated from bridge structures. In the first half of the movie the STA map is shown at low contour level to visualize parts of the ER membrane (top) and the OMM (bottom). In the second half of the movie, the contour level is increased. The scale bar is 5 nm. Note that due to the perspective view, the scale bar applies only to the front of the scene.

Supplementary Video 2

Segmentation model of ER–mitochondria contact site, imaged by cryo-ET at location of Mdm34–mNeonGreen. The membrane segmentation is the same as shown in Extended Data Fig. 2a. In addition, the STA map was placed at the positions of individual bridge structures (shown in green). The ER is shown in cyan (semi-transparent), OMM in yellow, IMM in mustard. Scale bar is 100 nm. Note that due to the perspective view, the scale bar applies only to the front of the scene.

Supplementary Video 3

Segmentation model of ER–mitochondria contact site, imaged by cryo-ET at location of Mdm34–mNeonGreen. The membrane segmentation is the same as shown in Extended Data Fig. 2b. In addition, the STA map was placed at the positions of individual bridge structures (shown in green). The ER is shown in cyan (semi-transparent), OMM in yellow, IMM in mustard, peroxisome in pink. Scale bar is 100 nm. Note that due to the perspective view, the scale bar applies only to the front of the scene.

Supplementary Video 4

Segmentation model of ER–mitochondria contact site, imaged by cryo-ET at location of Mdm34–mNeonGreen. The membrane segmentation is the same as shown in Extended Data Fig. 2c. In addition, the STA map was placed at the positions of individual bridge structures (shown in green). The ER is shown in cyan (semi-transparent), OMM in yellow, IMM in mustard. Scale bar is 100 nm. Note that due to the perspective view, the scale bar applies only to the front of the scene.

Supplementary Video 5

Segmentation model of ER–mitochondria contact site, imaged by cryo-ET at location of Mdm34–mNeonGreen. The membrane segmentation is the same as shown in Extended Data Fig. 2d. In addition, the STA map was placed at the positions of individual bridge structures (shown in green). The ER is shown in cyan (semi-transparent), OMM in yellow, IMM in mustard. Scale bar is 100 nm. Note that due to the perspective view, the scale bar applies only to the front of the scene.

Supplementary Video 6

Segmentation model of ER–mitochondria contact site, imaged by cryo-ET at location of Mdm34–mNeonGreen. The membrane segmentation is the same as shown in Extended Data Fig. 2e. In addition, the STA map was placed at the positions of individual bridge structures (shown in green). The ER is shown in cyan (semi-transparent), OMM in yellow, IMM in mustard. Scale bar is 100 nm. Note that due to the perspective view, the scale bar applies only to the front of the scene.

Supplementary Video 7

Segmentation model of ER–mitochondria contact site, imaged by cryo-ET at location of Mdm34–mNeonGreen. The membrane segmentation is the same as shown in Extended Data Fig. 2f. In addition, the STA map was placed at the positions of individual bridge structures (shown in green). The ER is shown in cyan (semi-transparent), OMM in yellow, IMM in mustard, peroxisomes in pink. Scale bar is 100 nm. Note that due to the perspective view, the scale bar applies only to the front of the scene.

Supplementary Video 8

Model of the ERMES heterotrimeric Mmm1–Mdm12–Mdm34 complex, obtained by integrative modelling. FoldDock prediction was followed by MDFF into the STA map. First, the STA map (grey) and a space-filling representation of the heterotrimeric complex are shown, before they sequentially fade and a ribbon representation of the heterotrimeric complex remains. Mmm1 is shown in orange, Mdm12 in purple and Mdm34 in green.

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Wozny, M.R., Di Luca, A., Morado, D.R. et al. In situ architecture of the ER–mitochondria encounter structure. Nature 618, 188–192 (2023). https://doi.org/10.1038/s41586-023-06050-3

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