Nuclear pore complexes (NPCs) fuse the inner and outer membranes of the nuclear envelope. They comprise hundreds of nucleoporins (Nups) that assemble into multiple subcomplexes and form large central channels for nucleocytoplasmic exchange1,2. How this architecture facilitates messenger RNA export, NPC biogenesis and turnover remains poorly understood. Here we combine in situ structural biology and integrative modelling with correlative light and electron microscopy and molecular perturbation to structurally analyse NPCs in intact Saccharomyces cerevisiae cells within the context of nuclear envelope remodelling. We find an in situ conformation and configuration of the Nup subcomplexes that was unexpected from the results of previous in vitro analyses. The configuration of the Nup159 complex appears critical to spatially accommodate its function as an mRNA export platform, and as a mediator of NPC turnover. The omega-shaped nuclear envelope herniae that accumulate in nup116Δ cells3 conceal partially assembled NPCs lacking multiple subcomplexes, including the Nup159 complex. Under conditions of starvation, herniae of a second type are formed that cytoplasmically expose NPCs. These results point to a model of NPC turnover in which NPC-containing vesicles bud off from the nuclear envelope before degradation by the autophagy machinery. Our study emphasizes the importance of investigating the structure–function relationship of macromolecular complexes in their cellular context.
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The three cryo-EM maps associated with this manuscript have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-10198, EMD-10660 and EMD-10661. The integrative models of S. cerevisiae NPC are available at Zenodo at https://doi.org/10.5281/zenodo.3820319 and the PDB-Dev database under accession numbers PDBDEV_00000051, PDBDEV_00000052 and PDBDEV_00000053. Unprocessed western blots are available in Supplementary Fig. 1. Because of their size, original imaging data are available from the corresponding author upon request.
The input data and the scripts used for modelling are available at Zenodo at https://doi.org/10.5281/zenodo.3820319.
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We thank J. Baumbach for critical reading of the manuscript; the members of the Beck and Mahamid laboratories and M. Schorb for invaluable input and support; D. Thaller, P. Lusk and E. Hurt for critical discussions and for providing S. cerevisiae strains; J. Sun for help in tomographic segmentation; and W. Wriggers for help with the Situs software. We thank Leica for a collaboration to develop the prototype cryo-confocal. We acknowledge support by EMBL’s electron microscopy core facility (EMCF) and IT Services. M.A. was funded by an EMBO a long-term fellowship (ALTF-1389–2016); J.M. received funding from the European Research Council (ERC 3DCellPhase- 760067); H.K.H.F. is supported by a fellowship from the EMBL Interdisciplinary (EI3POD) programme under Marie Skłodowska-Curie Actions COFUND (664726); B.P. acknowledges funding by Max Planck Society; F.W. was supported by an EMBO Long-Term Fellowship ALTF 764-2014; and M. Beck acknowledges funding by EMBL, the Max Planck Society and the European Research Council (ComplexAssembly 724349).
The authors declare no competing interests.
Peer review information Nature thanks James Hurley and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Fig. 1 In-cell structure of the S. cerevisiae NPC vs detergent-extracted NPC (EMD-7321).
a, Gold standard FSCs of in-cell cryo-EM map of the S. cerevisiae NPC. All the curves (nuclear ring NR, cytoplasmic ring CR, inner ring IR) intersect the 0.143 criterium at around 25 Å resolution. b, Local resolution analysis59 with colour-coded bar. c, In-cell cryo-EM map (grey, average of n = 250 NPCs) in comparison to cryo-EM map of detergent-extracted S. cerevisiae NPCs (blue, EMD-7321 at the suggested contour level) show significant differences in diameter and in interpretable features. The nuclear membranes and the Y-complexes are clearly discerned in the in-cell cryo-EM map, in contrast to EMD-7321. d, Tomographic slices through the maps shown in c at the level of the cytoplasmic (CR) and inner rings (IR) and an individual spoke (Sp). Lines in c indicate slicing position shown in d. Arrowheads indicate blurred features in the outer rings. Scale bar: 50 nm.
Extended Data Fig. 2 Systematic fitting of inner and outer ring components into the S. cerevisiae NPC map.
Each row shows the visualization of the top fits (left), the histogram of raw scores (middle), and a plot of the top five P values (right). In the P value plots, the statistically significant fits are coloured red (P < 0.05; P values were calculated using the two-sided test as implemented in fdrtool R-package, see Methods). All P values were adjusted for multiple comparisons using Benjamini-Hochberg procedure. The top fits are indicated in the histograms with an arrow and the score value. The number of sampled fits used to calculate P values after clustering of similar solutions was 14,348, 1,015, 1,039, 1,479, 1,354, and 1,183 for the rows from top to bottom. For the IR, the integrative model of the single spoke of the IR15 was used as the fitted structure. For the outer rings (CR and NR), the crystal structure of the yeast Y-complex was fitted or its parts corresponding to subcomplexes. The structures were fitted by an unbiased global search using UCSF Chimera60 and scored using the cross-correlation score about the mean as explained in the Methods. The IR complex was fitted to the entire spoke map, while the other structures were fitted to individual CR and NR segments.
Extended Data Fig. 3 Architectural model of S. cerevisiae NPC.
a, Comparison of fitted (to the S. cerevisiae NPC map from this study, depicted in grey) integrative inner ring complex models (red ribbons) from15 with 20 nm diameter difference. b, Representative integrative structural models of CR and NR Y-complex (blue ribbons) built in this work (see Methods) shown with the integrative model of P-complex (yellow ribbons) and refined IR (red ribbons) from15 fitted to the allocated density of the in-cell map (grey density) from this study. The Y-complexes are more extended as compared to reference15 version by ~40 Å. c, Representative integrative structural models of CR and NR Y-complex (blue ribbons), P-complex (yellow ribbons) and IR (red ribbons) from15 (PDB-Dev ID: PDBDEV_00000010) fitted to the S. cerevisiae NPC map (grey density) from this study, with respect to spatial reference frame from15. d, Representative integrative structural model of NR Y-complex (blue ribbons) built in this work shown together with the integrative model of the refined IR (red ribbons) from15 fitted to the allocated density of the S. cerevisiae NPC map (grey density). The localization probability densities of Mlp1, Mlp2 and Mlp1/Mlp2 ensemble from15 are displayed in orientation relative to the NR Y-complex as in15. Local mask refinement of n = 250 NPCs recovers extra densities in the NR that locate around the Nup120-Nup145C junction and the Nup107-Nup133 stem (left dark dotted frame). Most likely these densities account for parts of the basket since it resembles a filamentous structure15. It anchors to the regions of the Y-complex that crosslink to Mlp1 or Mlp215 and connects to the Nup85-Seh1 arm as proposed by the previous model15, but contrary to15, the filamentous region centres at the Y-complex vertex rather than at the Nup85 arm. On the right panel residues crosslinking to Mlp1 or Mlp215 are shown in sphere representation and coloured according to their confidence score (from15) with yellow – P < 0.01, green – P < 0.1, magenta P > 0.1. The shown residues, together with the yet unmodelled crosslink to residue 2 of Nup8515, emphasize that the highest confidence crosslinks are associated to the Y-complex, while the lowest confidence ones are associated to the IR. A second density (right dark dotted frame) remains unassigned, being proximate to residues crosslinking with Nup116, Nup100, Nup145N (see Extended Data Fig. 5c).
Extended Data Fig. 4 Validation of the orientation of the Nup159 complex.
a, Systematic fitting of the negative stain map of the Nup159 complex (yellow) into the S. cerevisiae NPC map (grey) using UCSF Chimera60 and Colores program from the Situs package61. As a negative control, also the mirror image of the negative stain map was fitted but did not lead to significant scores, further underlining the unambiguous nature of the fits. Each row shows the visualization of the top fits (left), the histogram of raw scores (middle), and a plot of the top five P values (right). In the P value plots, the statistically significant fits are coloured red (P < 0.05; P values were calculated using the two-sided test as implemented in fdrtool R-package, see Methods). All P values were adjusted for multiple comparisons using Benjamini-Hochberg procedure. The top fits are indicated in the histograms with an arrow and the score value. The number of sampled fits used to calculate P values after clustering of similar solutions was 585, 599, and 2243, for top, middle, and bottom rows respectively. b, Representative integrative Nup159 complex model from15 inside the in-cell S. cerevisiae NPC map (grey mesh) in the orientation determined in this work (left) versus the previously published orientation (right). The Nup159 complex model is shown in orange ribbons within yellow localization probability density from15 locally fitted with UCSF Chimera60. The previous orientation was reproduced by first fitting the entire model from15 to the in-cell cryo-EM map and then locally fitting the Nup159 complex to the density (which was needed to bring the Nup159 complex into the density and preserve the orientation). The dashed grey line indicates the flipping axis between the two fits. c, Superimposition of the crystal structure 3PBP27 onto the Nup82 β-propellers from the representative integrative Nup159 complex model from15 in the revised orientation predicts the position of Nup116, as confirmed by our knockout study (Fig. 2c). d, Visualization of two of the top resulting systematic fits of the 3PBP crystal structure into the cryo-ET map presented in this study confirms our nup116Δ structure (Fig. 2c). e, Crosslinks between the Nup159 complex and the Y-complex from15 support the new orientation (left) compared to the published orientation (right). Satisfied and violated crosslinks are depicted as blue and red bars respectively while the Nup159 complex and the Y-complex from15 are depicted within the relevant localization probability densities from15.
Extended Data Fig. 5 Nup116 positioning and possible Nup188 SH3-like domain interactions.
a, New positioning of the Nup116 versus b, previously published integrative model (PDB-Dev ID: PDBDEV_00000010 from15, right). The two S. cerevisiae NPC models were superimposed such that the IRs are aligned to the same reference frame. The Nup116 position is shown either as the density assigned to Nup116 based on the Nup116 knockout structure in the CR (a) or as localization probability densities retrieved from reference15 model (b). The major structural elements of the NPC are indicated. The Nup116 connector cable in the in-cell model (a) has been taken from reference15 based on its position relatively to the IR. Blue bars represent crosslinks from Nup116 to other Nups15. For the in-cell model, the cryo-ET map is displayed; for the model from15, the localization probability densities (not an EM map) are shown instead. c, SH3-like domain (magenta ribbons and dotted frames) of Nup18828 (yellow ribbons) bridges the interactions between the inner and the outer rings. The positions of Nup188 crosslinking to Nup116 (the number of times that some Nup188 residues crosslink with more than one Nup116 residues are denoted in the labels in parenthesis), Nup100 and Nup145N are indicated in sphere representation (red spheres) and their location suggests that they link the connecting interfaces between the IR and the outer rings: NR brown dotted frame corresponds to an unassigned density, CR green dotted frame corresponds to Nup116 density, NR green dotted frame corresponds to a second Nup116 density, see Extended Data Fig. 6c.
Extended Data Fig. 6 nup116Δ NPC structure and nuclear envelope remodelling.
a, In-cell structure of nup116Δ NPC cut in half along the central axis, at 25 °C on the left and at 37 °C on the right. At 25 °C diameter and general dimensions are alike the WT structure of Fig. 1b. The red dashed square indicates the position of the missing densities at neck region corresponding to Nup116 (see also Fig. 2c and Extended Data Fig. 5a). b, FSCs of nup116Δ NPC at 25 °C after masking the three rings (NR, IR, CR respectively nuclear, inner and cytoplasmic ring). The curves intersect 0.143 at around 35 Å resolution. On the right FSC of nup116Δ NPC at 37 °C calculated with a mask enclosing both the IR and the NR. The average was performed with a pixel size of 13.8 Å (original pixel size 4 times binned). The resolution is ~50 Å. c, Structural differences in the NR of the S. cerevisiae NPC from WT (left), and nup116Δ cells at the permissive (25 °C, middle) and non-permissive (37 °C, right) temperatures. The grey envelope represents the overlay of EM maps generated with two different masks – one enclosing the entire NR and IR and the second centred at the region of the extra densities around the Y-complex. The regions of the major differences are indicated with dashed frames. A shift of the Nup85 arm away from the IR is indicated with a black dashed arrow. The difference density around the Nup85 arm between the NPC maps from nup116Δ 25 °C and WT cells is shown in the inset in red and indicates a putative nuclear copy of Nup116 and the conformational shift of the Nup85 arm. Due to the low resolution, the IR of the nup116Δ 37 °C is shown as segmented densities assigned based on the fitting of the outer nuclear copy of the IR subcomplex. Question marks indicate predicted assignments based on similarity to the WT map but with a poor density insufficient for fitting. The HideDust tool of UCSF Chimera60 was used for clarity. d, Cryo-tomographic slice of nuclear envelope with inner nuclear membrane evaginations marked with asterisks in nup116Δ NPC at 25 °C. The histograms show that the number of inner nuclear membrane evaginations is significantly higher in the nuclear envelope of the mutant cells (32 in n = 145 cryo-tomograms, 42 lamellae) in comparison to WT envelopes (1 in n = 240 cryo tomograms, 100 lamellae); centre values represent the mean and error bars the SD; P < 0.0001 (Mann–Whitney test, two-tailed); N marks the nucleus and C marks cytoplasm, scale bar: 100 nm. e, Box plot showing the increase in nuclear size in nup116Δ cells in comparison to WT cells (~2.5-fold increase in volume) and a 4 to fivefold increase in comparison to WT upon shift to 37 °C. Light microscopy data of Nup84-eGFP, shown in Fig. 3c, were used to quantify the difference in nuclear volume as explained in the methods section. n = 100 nuclei were measured for each strain from four independent biological replicates (filled circles represent averages of the independent biological replicates). The statistical significance was evaluated with by one-way ANOVA with Dunn’s multiple comparisons test; centre values represent the mean and error bars the SD; ***P < 0.0002, **** P < 0.0001). f, Cartoon model depicting the summary of the results coming from Fig. 3c, d, e and Extended Data Fig. 6d, e. NPC are represented as black cylinders, NPC-concealing herniae as black circles, a nuclear membrane evagination is shown as semicircle.
Extended Data Fig. 7 NPC degradation and nuclear envelope reshaping upon starvation.
a, western blot and quantification of the degradation of Nup133, Nup188 upon Nitrogen starvation at 25 °C and 30 °C in WT and nup116Δ strain. eGFP was measured using anti-eGPF immunoblotting. eGFP′ denotes vacuolar eGFP remnant. Dpm1 was used as a loading control. Centre values represent the mean and error bars the s.d. of n = 3 biologically independent replicates. At 30 °C, Nup133, Nup188 are degraded fourfold less in comparison to WT. b, Typical tomographic plastic section of WT, atg8Δ S. cerevisiae nuclei after 0h of starvation (negative control of Fig. 4c) or of vps4Δ S. cerevisiae nuclei after 0 or 24 h of starvation. Quantification of number of NPC-exposing herniae at 0, 5 and 24 h of starvation is shown for vps4Δ cells. We show an example of a round nucleus and of an NPC-exposing hernia from those cells. n = 85 tomograms of nuclei for WT; n = 84 for vps4Δ; n = 83 for atg8Δ; n = 62 for vps4Δ 24h starved; n = 64 for vps4Δ 5h starved; see Supplementary Table 2. N marks the nucleus. Scale bar, 200 nm. c, Quantification of of NPC-exposing herniae from plastic section tomograms as in Fig. 4c at 5h and 24h of starvation. After 24h, 82% of nuclei in the atg8Δ cells contain NPC-exposing herniae (n = 78 tomograms in average for each condition, see Supplementary Table 2). d, Quantification of deconvoluted wide-field maximum intensity projection images as in Fig. 4d from three biologically independent replicates and n = 300 per replica. e, Quantification of NPC density (NPCs/μm2) from plastic sections tomograms as in c shows clustering of NPCs in NPCs-exposing hernia. The statistical significance was evaluated with one-way ANOVA with Dunn’s multiple comparisons test (n = 20 tomograms per strain except vps4Δ NPC-exposing herniae where n = 5 tomograms; the average nuclear envelope surface measured per strain is 20 μm2, except atg8Δ NPC-exposing herniae where is 11 μm2 and vps4Δ NPC-exposing herniae where is 3.5 μm2; ****P < 0.0001; centre values represent the mean and error bars the SD). f, Phase contrast and deconvoluted wide-field max intensity projection of WT, vps4Δ and atg8Δ cells imaged live before and after 24h of starvation with eGFP-tagged Nup192 as marker. The quantification is derived from three independent biological replicates where five images with at least n = 250 nuclei per replicate; central bars represent the mean and error bars the s.d. The trend of nucleus deformation is the same seen in b. g, Same as c for WT and atg8Δ cells, but with the inner nuclear membrane marker Nsg1. Data are derived from three independent biological replicates with at least n = 250 nuclei per replicate.
Extended Data Fig. 8 Interaction Nup159-Atg8 upon starvation.
a, Fluorescence intensity analysis of nup120Δ, Nup170-Mars, Nup159-Atg8-split Venus nuclei spots before and after 5.5 h of starvation. The ratio Mars/Venus shows the significant increase in Venus signal as compared to Mars signal in this starved strain. Three biological replicates and n = 100 spots per replicate were measured (P < 0.00001, Mann–Whitney, two-sided; box centres represent the mean and error bars the s.d.). b, Tomographic slice (plastic section) overlaid with wide-field fluorescent image obtained by on section-CLEM37,45 of the strain in a upon ~6 h of starvation. n = 36 correlated tomograms (technical replicates) were acquired and 24 (75%) had similar results. 8 spots come from uranyl autofluorescence in the cytoplasm. Venus signal is shown in yellow. Nup170-Mars is shown in red. The cyan dashed rectangle indicates the area zoomed into the two right panels showing tomographic slices at two different Z-heights (N marks the nucleus, scale bar: 200 nm). See also Supplementary Videos 9, 10.
Extended Data Fig. 9 Model of membrane remodelling in NPC-concealing and NPC-exposing hernia.
a, Plausible inside-out assembly intermediate33 or inner nuclear membrane evaginations (Extended Data Fig. 6d) progress into b, NPC-concealing herniae (Fig. 3a, Supplementary Video 3) which accumulate over time (Fig. 3d), concomitantly with increasing nuclear size (Extended Data Fig. 6e;3). Black lines indicate dark material present in cryo tomograms, possibly poly-A mRNA3. a matures into c, fully assembled NPCs as structurally analysed in cells frozen in exponential growth phase (Fig. 1b, c, Extended Fig. 3d). d, When autophagy is triggered by nitrogen starvation, NPCs cluster at the NPC-exposing hernia as in atg8Δ (Fig. 4c, d, Extended Data Fig. 7c–e, Supplementary Video 6). e, NPC clustering allows high avidity between Atg8 and Nup159 (Fig. 4b, Extended Data Figs. 7e, 8a, b, Supplementary Videos 5, 8, 9, 10) causing nuclear envelope budding depicted in f. f, Autophagosomes harbouring NPC-containing nuclear vesicles (Fig. 4e, Supplementary Video 7) are transported through the cytosol for degradation in the vacuole (g as in Fig. 4f). Colour-code as in Fig. 4e except that nuclear envelope membranes are depicted pink. Ribosomes are depicted as red circles; nuclear material as blue circles.
Supplementary Figure 1
Raw images of Western blots related to Extended Data Fig. 7a.
Supplementary Table 1
S. cerevisiae strains used in this study.
Supplementary Table 2
Tomograms acquired in this study.
Overview of the integrative model of Sc NPC from WT cells. The structural model is shown in cartoon representation, coloured by Nup identity, and shown fitted to the cryo-EM map (gray). Negative stain map of the Nup159 complex is shown as yellow surface fitted into the cryo-EM map.
Overview of the integrative model of Sc NPC from nup116Δ cells grown at 25 °C. Representation and colours as in Video 1.
Cryo electron tomogram of NPC-concealing hernia (nup116Δ after shifting 4h to 37 °C). Herniae of different sizes are present in this tomogram. Small herniae are highlighted by white circles. N marks the nucleus and C cytoplasm. n = 70 tomograms were acquired of this strain from grids prepared in different days from different cell cultures (biological replicates), see also Fig. 3a. Scale bar: 100 nm.
Overview of the integrative model of Sc NPC from nup116Δ cells grown at 37 °C. Representation and colours as in Video 1.
3D cryo-CLEM tomogram of Nup159-Atg8 interaction. The video shows NPC-exposing herniae in nup120Δ after 5,5h starvation. Clustered NPCs depicted in red are surrounded by double membranes depicted in yellow, see also Fig. 4b. n = 2 tomograms were acquired from grids prepared in two different days from two different cell cultures (biological replicates), the second tomogram is shown in Video 8. Scale bar: 100 nm.
NPC-exposed hernia in atg8Δ after 24h of starvation. Tomogram of atg8Δ S cerevisiae nuclei after 24h of starvation. See also Fig. 4c. n = 62 tomograms (technical replicates) were acquired. Scale bar: 100 nm.
NPC-containing nuclear vesicle in the cytoplasm. The vesicle is found in S cerevisiae atg15Δ cells starved ~24h. It is surrounded by cytosol content (ribosomes) and two membranes. Nuclear content is in cyan, nuclear membrane in yellow, double membrane in green. n = 5 tomograms acquired from grids prepared in different days from different cell cultures, see also Fig. 4e. Scale bar: 200 nm.
3D cryo-CLEM tomogram of Nup159-Atg8 interaction. NPC-exposing herniae are seen in nup120Δ cells after 5.5h starvation. Clustered NPCs depicted in red are surrounded by double membranes depicted in yellow, see also Fig. 4b. n = 2 tomograms were acquired from grids prepared in two different days from two different cell cultures, the second tomogram is shown in Video 5. Scale bar: 100 nm.
Plastic CLEM tomogram of Nup159-Atg8 interaction. NPC-exposing herniae in nup120Δ cells after 5.5h starvation. Clustered NPCs depicted in red are surrounded by double membranes depicted in yellow. n = 24 correlated tomograms (technical replicates) were acquired similar to this video, see also Video 10. Scale bar: 100 nm.
Plastic CLEM tomogram of Nup159-Atg8 interaction. NPC-exposing herniae in nup120Δ cells after 5.5h starvation. Clustered NPCs depicted in red are surrounded by double membranes depicted in yellow. n = 24 correlated tomograms (technical replicates) were acquired similar to this video, see also Video 9. Scale bar: 100 nm.
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Allegretti, M., Zimmerli, C.E., Rantos, V. et al. In-cell architecture of the nuclear pore and snapshots of its turnover. Nature 586, 796–800 (2020). https://doi.org/10.1038/s41586-020-2670-5
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