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A combined biochemical and computational approach provides evidence for membrane remodelling by the structural scaffold of the endocytic TPLATE complex

Nature Plants volume 11, pages 2423–2436 (2025)Cite this article

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Abstract

Eukaryotic cells maintain homeostasis of their outer membrane by controlled internalization of lipid and protein constituents via endocytosis. Endocytosis is evolutionary conserved and uses similarly folded domains. How these structural folds are combined into proteins and protein complexes, however, differs between eukaryotic kingdoms. The TPLATE complex (TPC) in plants is an evolutionary ancient protein module that combines several protein domains with a conserved role in endocytosis into a single octameric protein complex. Its molecular architecture, lipid-nucleated condensate formation and requirement for clathrin cage curvature revealed its function in endocytosis initiation in plants. Mechanistic understanding of how this complex drives membrane deformation during plant endocytosis is, however, lacking. Here we used an integrative structural approach to obtain a precise molecular structure of the TPC of Arabidopsis thaliana. In addition, our approach allowed visualizing the structural flexibility that hallmarks this enigmatic complex. We prove that the intrinsic structural flexibility is required for its functionality and membrane recruitment. The membrane-binding interface consists of several domains with differential lipid preferences. Finally, we demonstrate via molecular dynamics simulations that the crescent shape of the structured part of the complex is sufficient for membrane curvature generation. Our mechanistic insight, obtained by a combined biochemical and computational approach, shows that the structured part of the TPC likely contributes to the execution of plant endocytosis, which does not depend on cytoskeletal-based force generation.

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Fig. 1: The structure of the TPC is determined via integrative modelling.
Fig. 2: TPC positions structural domains intermediate to both AP-2 and COPI.
Fig. 3: TPC establishes domain-specific lipid interactions.
Fig. 4: TPC function requires structural flexibility of the TML μHD.
Fig. 5: The structured domains of TPC provide the force for membrane deformation.

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Data availability

The nsEM map calculated from the full L-arginine dataset and used as a base for the integrative modelling is deposited in the EMDB database (EMD-54584). All data files related to integrative modelling, molecular dynamics simulations and the electron microscopy maps calculated to confirm the map deposited at EMDB (split L-arginine and creatine datasets) are available via Zenodo at https://doi.org/10.5281/zenodo.16362990 (ref. 93). The mass spectrometry data are deposited at the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD056680. Source data are provided with this paper.

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Acknowledgements

We thank A. De Meyer and J. M. Dragwidge for help with live cell imaging, F. Delaere and F. Coppens for the access and maintenance of the server and J. Nolf for help with Äkta-based protein purification. We thank F. Impens and A. Staes for help with XL-MS and S. Savvides, J. Felix and K. Verstraete for their help with the Cryo plunger and with the CryoSparc software package. We also thank R. Efremov for constructive discussions on the purification and imaging strategies, and H. Martinez-Seara Monne for feedback on the membrane bending force. This work was supported by the European Research Council Grant T-REX 682436 (D.V.D.), the Research Foundation – Flanders (FWO) G017919N (D.V.D.), The Czech Science Foundation grant number 22-35680M (R.P.) and project GA UK number 154324 (M.N.). Computational resources used for molecular dynamics simulations were provided by the e-INFRA CZ project (ID: 90254), supported by the Ministry of Education, Youth and Sports of the Czech Republic.

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  1. These authors contributed equally: Julia M. Kraus, Michaela Neubergerová.

Authors and Affiliations

  1. Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium

    Julia M. Kraus, Alvaro Furones Cuadrado, Neeltje Schilling, Dominique Eeckhout, Nancy De Winne, Eveline Van De Slijke, Michaël Vandorpe, Klaas Yperman, Evelien Mylle, Geert De Jaeger & Daniël Van Damme

  2. VIB Center for Plant Systems Biology, Ghent, Belgium

    Julia M. Kraus, Alvaro Furones Cuadrado, Neeltje Schilling, Dominique Eeckhout, Nancy De Winne, Eveline Van De Slijke, Michaël Vandorpe, Klaas Yperman, Evelien Mylle, Geert De Jaeger & Daniël Van Damme

  3. Institute of Experimental Botany of the Czech Academy of Sciences, Prague, Czech Republic

    Michaela Neubergerová & Roman Pleskot

  4. Department of Experimental Plant Biology, Faculty of Science, Charles University in Prague, Prague, Czech Republic

    Michaela Neubergerová

  5. VIB-VUB Center for Structural Biology, Brussels, Belgium

    Marcus Fislage

  6. Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium

    Marcus Fislage

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  1. Julia M. Kraus
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Contributions

J.M.K., M.N., A.F.C., N.S., M.V., K.Y. and E.M. performed experiments. N.D.W. and E.V.D.S. generated PSB-D cell cultures. R.P. performed integrative modelling. D.E. performed mass spectrometry data analysis. M.F. helped with electron microscopy data recording and processing. G.D.J., R.P. and D.V.D. designed experiments and supervised the work.

Corresponding authors

Correspondence to Roman Pleskot or Daniël Van Damme.

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Nature Plants thanks Jianzhong Liu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 TPC purification and negative stain structure generation.

a) Stain free SDS PAGE gels showing the purification progress of TPC from Arabidopsis PSB-D cell suspension culture. The TEV elution during the StrepTag purification elutes TPC well as there is no residual complex on the beads (Strep B). Since the HIS-tag site is not part of the bait, but part of TPC, excessive bait cannot bind the Ni-NTA Sepharose beads (IMAC FT). The established purification conditions allow us to elute TPC selectively while retaining the TEV on the beads (IMAC B & IMAC E), yielding a clean TPC sample. b) To identify the individual subunits, purified TPC was analyzed via Western blots, using antibodies against selected TPC subunits (black font), namely: anti-TWD40-2, anti-TASH3, anti-AtEH1, anti-TPLATE and anti-LOLITA. As described before, the anti-AtEH1 antibody cross reacts with AtEH2 visible by the second band in the Western blot. Both anti-TASH3 and anti-LOLITA are antibodies developed as part of this research and the specificity is presented in Extended Data Fig. 10. The annotation via Western blot was complemented by single band SDS page MS experiments. These results confirmed the Western Blot analysis and allowed the annotation of the remaining subunits (grey font) (Source file). c) The 3D map generated with the full L-arginine dataset (grey) was validated by splitting the dataset into two parts and a 3D map was reconstituted for each part individually (part 1 = orange, part 2 = green). In addition to that, a 3D map was reconstituted from TPC purified in a creatine containing buffer (magenta). These independently calculated maps were then compared via UCSF ChimeraX against the map from the full L-arginine dataset. The correlation value of these pairwise comparisons is depicted next to the corresponding map. As is visible in c), the maps all present the same shape and dimensions. The scale bar measures 50 Å. d)-i) show the 3D reconstruction of the L-arginine dataset. d) After 3 rounds of 2D classification, where particles were discarded, we yielded a final particle set of 156 997 particles. 2D classes corresponding to this final set are depicted in d). e) To exclude that good particles were removed during 2D classification, all discarded particles were re-classified. The resulting 2D classes are presented in e) and no 2D classes of interest were present. f) The densities plotted in the real space slices indicate that TPC particles can be clearly distinguished from the background and noise of the micrographs during 3D refinement. g) The Fourier shell correlation (FSC) estimates a 17 Å resolution at 0.5 FSC for the refined 3D map. h) The mask that was determined during the 3D refinement to compute the 3D map is in accordance with the real space slices in panel f). i) The Azimuth graph positions all particles used for the 3D refinement onto the refined 3D TPC map. This results in a plot where one can determine which view angle has more, or less particles. Ideally one would have an even distribution over the whole graph. The uneven distribution visible in this graph indicates a preferred orientation of the TPC on the negative stain EM grid.

Source data

Extended Data Fig. 2 Integrative modeling of TPC and modeling validation.

a) The scheme illustrates different stages of the integrative modeling process, including data gathering, representation of the subunits, configurational sampling and model visualization. The input data included the negative stain electron microscopy (nsEM) map, chemical cross-links (XL-MS), multiscale representation of the TPC subunits, TPC subcomplexes modeled by Alphafold2 and stereochemical restraints. b) Distance distribution of obtained chemical cross-links in the top-scoring cluster. The orange line represents the threshold for the consistent cross-links. No cross-link was violated in the top-scoring cluster. c) Convergence of the model score calculated for the ensemble of the good-scoring models. Scoring did not improve after the addition of more independent models. The error bars represent the standard deviation of the best scores, estimated by repeating the sampling 10 times. The red line depicts a lower bound on the total score. The error bars represent SD. d) Splitting of the good-scoring models into two sample populations (blue and light red) resulted in significantly different score distributions (two-sided Kolmogorov-Smirnov two-sample test, K-S test, p-value = 1.7e-32), but the magnitude of the difference is, however, negligible (0.07), therefore both score distributions are effectively equal. e) Determining the sampling precision. The sampling precision as defined by three criteria; first, the p-value calculated using the two-sided χ2-test for homogeneity of proportions (red dots); second, an effect size for the χ2-test is quantified by the Cramer’s V value (blue squares); third, sufficiently large clusters (containing at least 10 models) visualized as green triangles. The vertical dotted gray line indicates the root mean square displacement (RMSD) clustering threshold at which three criteria are satisfied (p-value > 0.05, Cramer’s V < 0.10, and the population of clustered models > 0.80). The calculated sampling precision is 40 Å. f) Using the sampling precision as the threshold, populations of sample 1 (light red) and 2 (blue) form eight clusters. 90.5 % of the models belong to cluster 0, which has a precision of 31.5 Å. g) Localization density maps for Sample 1 and Sample 2 of Cluster 0, visualized here at a threshold equal to one-tenth the maximum. The cross-correlation of the localization density maps of the two samples is 0.985, indicating that the position of TPC subunits in the two samples is identical.

Source data

Extended Data Fig. 3 Visualization of the high and low-precision regions of the integrative model.

a) Precision of the TPC integrative model as well as that of the individual subunits according to the PrISM method. b) Overall high-precision of the core sub-complex consisting of the TASH3 trunk domain, LOLITA, the TPLATE trunk domain and the TML longin domain. c) Precision of the TWD40 subunits arching the inner core. The structured parts are well resolved with the exception of the all alpha domain (alpha), which contains a flexible linker. d) Precision of the AtEH/Pan1 subunits. As these subunits present the most flexible subunits in the complex, they have the lowest precision in the integrative TPC model. e) - i) Compare the integrative models obtained by ref. 23 and obtained by this study. e) Comparison of the centroid structures. f) Comparison of the position of TPLATE and LOLITA relative to the TASH3 body. g) Comparison of the position of TPLATE and TWD40-1 relative to the TASH3 body. h) Comparison of the position of TWD40-1 and TWD40-2 relative to the TPLATE appendage. i) The rotation of the TPLATE body relative to the TASH3 body between the models is a key contributor to the difference in the arrangements depicted in f) - h).

Extended Data Fig. 4 Comparison between the integrative and the AlphaFold3 generated model of the TPC hexamer.

a) Ribbon representation of the truncated TPC hexamer obtained by integrative modeling and Alphafold3 (AF3). b) The matrix shows the Predicted Alignment Error (PAE) for the AF3 model of the truncated TPC hexamer. c) Inter- and intramolecular DSSO and BS3 cross-links were mapped on both models. The AlphaFold3 model mainly differs from the integrative structure in the position of the TASH3 trunk domain and LOLITA. The position of the TASH3 trunk domain and LOLITA in the Alphafold3 model is not supported by the identified cross-links. d) PAE plots for the AF3 models of two TPC subcomplexes containing EH subunits. The positions of TML µHD, EH1 and EH2 could not be confidently modeled. Upper PAE plot - TPC subcomplex composed of TPLATE, TML, TWD40-1, TWD40-2, N-terminal half of AtEH1/Pan1 and N-terminal half of AtEH2/Pan1. Lower PAE plot - TPC subcomplex composed of TWD40-1, TWD40-2, AtEH1/Pan1 and AtEH2/Pan1.

Extended Data Fig. 5 Conservation analysis for structure comparison.

a) ConSurf analysis for all COPI and AP-2 subunits present in A. th. A floating average of 5 residues was calculated from the per-residue ConSurf score. This average was then colored green, if the value was below 5 and the sequence therefore variable, or it was colored purple if the score was above 5 and therefore conserved. b) The AP-2β appendages are the least conserved domains in the analysis performed in a). To demonstrate the structural conservation for the AP-2β appendages, structural models of the Arabidopsis AP-2β subunits were taken from the AlphaFold2 database and the appendage domains were aligned to the published human cryo EM model (PDB 6YAI) with UCSF ChimeraX. The AP-2β appendages of both Arabidopsis alleles are structurally nearly identical to their human counterpart, as is evident from their RMSD values. c) To demonstrate the conservation of the disordered TML linker, we performed a ConSurf analysis for TML as in a). This conserved 210 residue linker was replaced with the shorter 38 residue long AP-2μ linker from Arabidopsis in the TML-AP-2μ chimera. The legend below c) applies to a) and c). The scale bar represents 250 residues.

Source data

Extended Data Fig. 6 LUVs of different lipid compositions do not tubulate without TPC.

a) - c) LUVs with no TPC added do not tubulate. The images are taken at 25 000 times magnification and the scale bars measure 500 nm. a) Depicts uncharged LUVs, while in b) LUVs contain 20 % PA and in c) the PI4,5P2 content is 9 %. n = number of acquired micrographs.

Extended Data Fig. 7 MD simulations reveal TPC membrane interactions.

a) Selected snapshots of MD simulation time series showing TPC interaction with the membrane. The minimum distance between TWD40-1, TWD40-2 and TASH3 and the membrane was calculated to monitor TPC-membrane interaction. The last 2 μs of trajectories in which TWD40-1, TWD40-2 and TASH3 simultaneously bind the membrane, were selected for follow up analyses. Phosphatidylcholine is depicted in black, phosphatidylethanolamine is depicted in dark grey, PS, PA, PI4P and PI4,5P2 are depicted in shades of blue. b) ZZ component of the rotational matrix visualizing rotation of the TPC in the simulation box in time. 4 out 5 simulation replicas show no rotation of the complex once bound to the membrane. Rzz = 1 no rotation, Rzz = 0 rotation 90°/270°, Rzz = -1 rotation 180°. c) Residues of the membrane-binding interface of the TPC hexamer interacting with phosphatidylserine (PS). d) PI4P, PI4,5P2, PA and PS densities around TPC hexamer. PI4P, PI4,5P2 cluster at the membrane-binding interface. e) Membrane curvature over the last 2 μs calculated for each MD trajectory. In 3 out of 5 simulation repeats, TPC interacting with the membrane induces the concave membrane curvature. The time indications represent computational time of CG-MD simulations, which cannot be easily compared to the actual endocytic life times observed in microscopy experiments.

Source data

Extended Data Fig. 8 Expression and imaging controls for plant-related experiments.

a) Expression analysis of TWD40-1-β-GFP in independent plant lines in Col-0 background that were used for imaging. The stain free gel that serves as loading control is shown on top. The anti-GFP Western blot shows that the constructs are stably expressed. The Western Blot was done once. b) Bleed through control images for the data shown in Fig. 3d. The left and middle panels show the signal of a Col-0 root using our imaging conditions at different LUT levels as well as the FM4-64 channel image. Our imaging conditions, using a combination of three excitation lines for GFP does not result in FM4-64 bleed through. The scale bars equal 25 μm. This analysis was done for six independent plants. c) Expression analysis of independent TML-GFP and TML-AP-2μ-GFP lines in tml-1 background. The Western blot indicates both constructs are expressed at comparable levels. The Western Blot was done once. d) To control for random associations of TML-GFP (top panels) or TML-AP-2μ-GFP (bottom panels) with TPLATE-mSCARLET in the colocalization analysis, the green channel was rotated by 90° to the right and the images were re-analyzed. The pie chart quantifies the colocalization observed. Gray = colocalization, magenta = TPLATE only and green is TML only. Scale bars equal 5 μm. Two independent lines per construct were used and three plants were analysed per line. Of each root, six cells were used for the quantification.

Source data

Extended Data Fig. 9 The structured domains of TPC provide the force for membrane deformation.

a) Side and top view images of representative timepoints from the MD simulation of a membrane without TPC. The 0 µs time point represents the starting point and the membrane is roughly 60 nm x 60 nm in size. At 5 µs, the membrane has essentially the same size. The height of the simulation box remains constant throughout the simulation, indicating that no membrane deformations occur. Different colors indicate different simulation repeats. b) Side and top view images of representative timepoints from the MD simulation of a membrane with a single TPC. The 0 µs time point represents the starting point and the membrane is roughly 40 nm x 40 nm in size. At 5 µs, the membrane has essentially the same size. The height of the simulation box remains constant throughout the simulation, indicating that no membrane deformations occur. Different colors indicate different simulation repeats. c) Visualization of the deformation of the simulation box over the time of the simulation for 3x TPC (Fig. 5g) extended up to 5 µs. The increased height of the simulation box (4 out of 5 simulation repeats) indicates membrane deformations. Different colors indicate different simulation repeats. This panel represents an extension of the simulation shown in Fig. 5g. d) Side and top view images of representative timepoints from the MD simulation of a membrane with a single AP-2. The 0 µs time point represents the starting point and the membrane is roughly 40 nm x 40 nm in size. At 5 µs, the membrane has essentially the same size. The height of the simulation box remains constant throughout the simulation, indicating that no membrane deformations occur. Different colors indicate different simulation repeats. e) Side and top view images of representative timepoints from the MD simulation of a membrane with 3x AP-2. The 0 µs time point represents the starting point and the membrane is roughly 60 nm x 60 nm in size. At 4.7 µs, the membrane is deformed. The increased height of the simulation box (3 out of 5 simulation repeats) indicates membrane deformations. Different colors indicate different simulation repeats. This panel represents an extension of the simulation shown in Fig. 5g. f) Side and top view images of representative timepoints from the MD simulation of a membrane with a single AP180 ANTH domain. The 0 µs time point represents the starting point and the membrane is roughly 40 nm x 40 nm in size. At 5 µs, the membrane has essentially the same size. The height of the simulation box remains constant throughout the simulation, indicating that no membrane deformations occur. Different colors indicate different simulation repeats. g) Side and top view images of representative timepoints from the MD simulation of a membrane with 3x AP180 ANTH domain. The 0 µs time point represents the starting point and the membrane is roughly 60 nm x 60 nm in size. At 5 µs, the membrane has essentially the same size. The height of the simulation box remains constant throughout the simulation, indicating that no membrane deformations occur. Different colors indicate different simulation repeats. This panel represents an extension of the simulation shown in Fig. 5g. h) Visualization of the membrane deformations along the Z axis (brown line and area) over the time alongside with the positions of the center of mass of the three TPLATE complexes (red, blue and green lines). Four MD simulation repeats that were not included in Fig. 5f are shown here. The time indications represent computational time of CG-MD simulations, which cannot be easily compared to the actual endocytic life times observed in microscopy experiments.

Source data

Extended Data Fig. 10 TASH3 and LOLITA antibody development.

a) and c) The pre-immunization sera of the rabbits were tested against Col-0 lysate to ensure the absence of background immunity. The Western Blot was performed once. b) The post-immune serum, as well as the purified anti-TASH3 antibody were tested against plant lysate of a GFP-TASH3 expressing plant, as well as against purified TASH3-FLAG. The serum generates a substantial amount of background signal. The purified anti-TASH3 antibody has less background signal in the plant sample, but it still recognizes multiple bands. It works well against purified samples. The specificity in the plant lysate was confirmed via anti-GFP (magenta arrow heads). The Western Blot was performed once. d) The post-immune serum of the anti-LOLITA antibody showed a substantial amount of background signal in plant lysates, whereas the purified antibody presented only little background signal. The purified antibody was not sufficient to detect native LOLITA in Col-0 lysate, however it could detect both purified LOLITA-HIS and LOLITA-GFP expressed in planta. The Western Blot was performed once. e) The specificity of the anti-LOLITA antibody was again confirmed against anti-GFP in the LOLITA-GFP expressing sample (orange arrow heads d & e). The Western Blot was performed once.

Source data

Supplementary information

Supplementary Information

Extended methods.

Reporting Summary

Supplementary Table 1

Primers, plasmids and plant lines.

Supplementary Table 2

GFP trap MS results.

Supplementary Table 3

XL-MS results.

Supplementary Table 4

SDS–PAGE single-band MS results.

Supplementary Table 5

Molecular dynamics parameters and force calculations.

Supplementary Video 1

Integrative modelling of the TPC structure.

Supplementary Video 2

Structured domains of TPC deform the membrane.

Source data

Source Data Fig. 1

Amino acid positions of the visualized crosslinks.

Source Data Fig. 3

Unprocessed western blots and quantification data.

Source Data Fig. 4

Statistical analysis and quantification data.

Source Data Fig. 5

Classification of Intrinsically Disordered Ensemble Regions (CIDER) analysis and simulation parameters.

Source Data Extended Data Fig. 1

Unprocessed western blots and statistical source data.

Source Data Extended Data Fig. 2

Amino acid position of crosslinks and statistical source data.

Source Data Extended Data Fig. 5

Consurf input data.

Source Data Extended Data Fig. 7

Simulation parameters.

Source Data Extended Data Fig. 8

Unprocessed western blots and quantification data.

Source Data Extended Data Fig. 9

Simulation parameters.

Source Data Extended Data Fig. 10

Unprocessed western blots.

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Kraus, J.M., Neubergerová, M., Cuadrado, A.F. et al. A combined biochemical and computational approach provides evidence for membrane remodelling by the structural scaffold of the endocytic TPLATE complex. Nat. Plants 11, 2423–2436 (2025). https://doi.org/10.1038/s41477-025-02146-y

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