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. 2018 Sep;561(7724):561-564.
doi: 10.1038/s41586-018-0526-z. Epub 2018 Sep 17.

Structure of the membrane-assembled retromer coat determined by cryo-electron tomography

Oleksiy Kovtun #  1   2 , Natalya Leneva #  3   4 , Yury S Bykov  1   2 , Nicholas Ariotti  3   5 , Rohan D Teasdale  3   6 , Miroslava Schaffer  7 , Benjamin D Engel  7 , David J Owen  8 , John A G Briggs  9   10 , Brett M Collins  11
Affiliations

Structure of the membrane-assembled retromer coat determined by cryo-electron tomography

Oleksiy Kovtun et al. Nature. 2018 Sep.

Abstract

Eukaryotic cells traffic proteins and lipids between different compartments using protein-coated vesicles and tubules. The retromer complex is required to generate cargo-selective tubulovesicular carriers from endosomal membranes1-3. Conserved in eukaryotes, retromer controls the cellular localization and homeostasis of hundreds of transmembrane proteins, and its disruption is associated with major neurodegenerative disorders4-7. How retromer is assembled and how it is recruited to form coated tubules is not known. Here we describe the structure of the retromer complex (Vps26-Vps29-Vps35) assembled on membrane tubules with the bin/amphiphysin/rvs-domain-containing sorting nexin protein Vps5, using cryo-electron tomography and subtomogram averaging. This reveals a membrane-associated Vps5 array, from which arches of retromer extend away from the membrane surface. Vps35 forms the 'legs' of these arches, and Vps29 resides at the apex where it is free to interact with regulatory factors. The bases of the arches connect to each other and to Vps5 through Vps26, and the presence of the same arches on coated tubules within cells confirms their functional importance. Vps5 binds to Vps26 at a position analogous to the previously described cargo- and Snx3-binding site, which suggests the existence of distinct retromer-sorting nexin assemblies. The structure provides insight into the architecture of the coat and its mechanism of assembly, and suggests that retromer promotes tubule formation by directing the distribution of sorting nexin proteins on the membrane surface while providing a scaffold for regulatory-protein interactions.

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Conflict of interest statement

The authors declare that they have no competing financial interests

Figures

Extended Data Figure 1
Extended Data Figure 1. The retromer-Vps5 complex in solution and binding to membranes
(a) Retromer forms a stable complex in solution. Fractions containing retromer (Vps35, Vps26 and Vps29) after gel-filtration on a Superdex 200 column analysed by Coomassie-stained SDS-PAGE. (b) Gel-filtration profile of Vps5 and MALLS analysis of molecular weight. Mean molecular weight and standard deviation from three independent gel-filtration experiments are shown. The expected molecular weight of Vps5 monomer is 67 kDa, so the observed molecular weight of 129 kDa indicates formation of a homodimer (c). A Coomassie-stained SDS-PAGE of Vps5 fractions from b. (d) Vps5 binds to retromer in solution. SDS-PAGE of GST-Vps5 and of retromer are given in the “input” panels. Retromer was incubated with GST-tagged Vps5 or GST baits, and the resultant complex was isolated on Gluthathione Sepharose beads (“pull-down” panels). The bottom panel shows the intact PAGE gel used to extract lanes for the upper panel. (e) Retromer membrane recruitment is dependent on Vps5. GST-Vps5 alone, GST-Vps5 with retromer complex, and retromer complex alone were incubated with liposomes and pelleted to isolate the liposome-bound protein fraction. Supernatant (S) and pelleted fraction (P) were compared with Coomassie stained SDS-PAGE. PC/PE liposomes were used as a negative control. Vps5 is efficiently pelleted by Folch brain extract liposomes, and the introduction of PI3P does not increase the amount of pelleted protein. The retromer complex shows no membrane association on its own, but is recruited to Folch and Folch/PI3P membranes when it interacts with Vps5. (f) Retromer promotes tubule formation by Vps5. Characteristic cryoEM images at medium (left) and high (right) magnification of Folch liposomes incubated either with Vps5 alone (top panels) or in the presence of the retromer complex (bottom panels). Data shown in all panels are representative of at least three independent experiments.
Extended Data Figure 2
Extended Data Figure 2. Overview of the subtomogram averaging procedure.
Stages in the subtomogram averaging procedure are shown from top to bottom. Key steps are illustrated by average volumes (grey) overlaid with the corresponding alignment mask (gold). Alignment masks are shown at 0.5 value threshold. Volumes in a-c are low-pass filtered to 50 Å. (a) The final iteration of reference-free subtomogram averaging procedure independently conducted in bin4 tomograms that were acquired at -2.5 mm (left) and -5.5 mm (right) defoci, filtered to 50 Å resolution. (b) The average of the references shown in a. (c) The volume from b was rotated to place either the apex of the arch (left) or the base of the arch (right) in the box centre, 2-fold symmetrized, and filtered to 50 Å. These two volumes were used as starting references for further alignments. (d) The references after alignment at bin8. (e) The references after alignment at bin2. After SA convergence in bin2, focused alignment was conducted on individual structural features. The final maps are shown in f1-f3 and f4-f5 for alignments focussed on the regions within the gold alignment masks.
Extended Data Figure 3
Extended Data Figure 3. CryoET map and reconstruction resolution.
(a) Mask-corrected FSC curves for each of the final focused maps shown in Extended Data Fig. 2f. The overall resolution at the 0.143 criterion is marked. (b) Sharpened maps coloured by local resolution according to the indicated colour map determined by FSC within a moving local mask. Arrowheads indicate an unassigned density, which may correspond to a helical element in loop 305-387 of Vps35.
Extended Data Figure 4
Extended Data Figure 4. Crystal structure of C. thermophilum Vps29, and comparison of cryoET structures with previous crystal structures.
(a) Crystal structure of C. thermophilum Vps29 (red) overlaid with the crystal structure of human Vps29 (blue). Crystallographic structure determination statistics are given in Extended Data Table 2. (b) The fitted Vps26 dimer model with monomers coloured in dark green and light green. The homodimeric interface is formed by β-sheet extension of two N-terminal β-sandwich domains. The positions of the docked Vps26 models suggest formation of an extended hydrophobic core between subunits. Close-up images of fitted Vps26 subunits highlight the extended hydrophobic core. (c) Surface representation of the Vps26-Vps35-Vps29 trimer mapped with binding regions for retromer effectors. Neighbouring Vps5 and retromer proteins in the assembled array are shown as ribbons. Retromer components are coloured as in Fig. 1. Lower panels show higher magnification views of the overviews in the upper panels. Binding sites observed in structural data are coloured according to colour of the corresponding label; dashed lines indicate binding regions identified in biochemical assays. The binding interfaces of human Snx27, Snx3, Snx3/Dmt1-II, Varp/TBC1d5 were modelled using coordinates with PDB accession numbers 4P2A, 5F0L and 5GTU respectively; the Dmt1-II cargo peptide is shown as a ribbon. The Snx3/Dmt1-II binding site overlaps with that of Vps5. The binding site of the Snx27 PDZ domain on Vps26 is accessible although due to a lack of structural information on full-length Snx27 it is unclear whether this binding is simultaneously consistent with membrane binding by the Snx27 PX domains. The regulatory factors Varp and TBC1d5 share a binding interface on Vps29 that is exposed towards the outer extremity of the coat. This site in human Vps29 is also hijacked by the RidL protein from the pathogen Legionella pneumophila,. However, as for Snx27, full-length structures of Varp, TBC1d5 and RidL are not available so we cannot be sure how they will be arranged in the fully assembled array. Rab7 has been speculated to contribute to membrane recruitment of retromer by binding to Vps35 in the region indicated by the dashed line,. The deletion of this helical region (helix 6 in S. cerevisiae) resulted in loss of interaction with Ypt7 (the Rab7 homologue in yeast). It has been shown recently that retromer binding to the PX-BAR complex displaces Rab7 during formation of tubules,. The Vps10 “binding site” (dashed line) indicates a region where point mutations affect Vps10 recycling, however, no biochemical interactions between Vps10 and retromer have been shown, and our efforts at detecting a physical interaction between Vps35 and the cytosolic domains of Vps10 have not shown any direct binding. (d) Snx3 and Dmt1-II (transparent yellow and dark green surfaces respectively), as bound to Vps35/Vps26 from PDB 5F0L, overlayed with our retromer-Vps5 complex structure (ribbons, coloured as above) demonstrating a sterical clash between Vps5 BAR and Snx3 PX domains. Note that C-terminal helix of Vps5 BAR (arrowheads) clashes with the Dmt1-II cargo peptide density. Left panel shows the same view as in the panel above in C; right panel shows the model rotated by 90 degrees around the vertical axis to provide the view along the long axis of BAR domain.
Extended Data Fig 5
Extended Data Fig 5. Global rigid body docking of Vps5, Vps26/Vps35(N) and Vps35(C)/Vps29, and adaptation of the retromer coat to different membrane curvatures.
(a-c) Fitting of structures to electron density maps was performed from 10000 random initial placements of atomic models using Chimera fit command. The cross-correlation between model and EM map, is plotted against the fraction of the structural model within the EM density threshold for: (a) Global fit of Vps5 dimer into the membrane-associated BAR domain density under the arch (map f3 in Extended Data Fig. 2); (b) Global fit of Vps26/Vps35(N) into the base of the arch (map f5 in Extended Data Fig. 2). (c) Global fit of Vps29/Vps35(C) into the apex of the arch (map f1 in Extended Data Fig. 2). Arrows indicate the high-scoring rigid body fits which were used as starting points for flexible fitting. (d, e) For a subset of ~50% of the data we calculated tube centroids by spline fitting, and determined local membrane curvature as the inverse of the distance from the subtomogram to the tube centroid. (d) Slices through averages of 20% of the subtomograms from the dataset with lowest (left) and highest (right) membrane curvature, focused on the arch (top) or Vps26-dimer (bottom). See also animation in Supplementary Video 5. (e) Distribution of membrane curvatures of retromer tubules in situ and in vitro. Lumenal diameter of each tube were measured manually from which mean and standard deviation were calculated.
Extended Data Figure 6
Extended Data Figure 6. Comparison of yeast PX-BAR proteins Vps5 and Vps17.
(a) Overlay of the Vps5 heterodimer model after flexible fitting into the cryoET structure (blue) with the human Snx9 PX-BAR domains (beige; the bound PI3P headgroup is also shown in magenta in stick representation). The PX and BAR domains in Vps5 adopt a very similar architecture to the Snx9 protein, but there are variations in the angle between the BAR domains, and in the orientations of the lateral PX domains. The second and third α helices of the Vps5 BAR domain are also longer than in Snx9. (b) Sequence alignment of Chaetomium thermophilum (CtVps5, CtVps17) and human (hSnx1, hSnx5) PX-BARs. CtVps5 secondary structure is indicated above the sequences. Sequence alignment and its representation were prepared in MultAlign and ESPript 3.0. (c) Overlay of ribbon models of CtVps5 (blue) and CtVps17 (grey). CtVps17 structure was modelled using CtVps5 as a template (SWISS-MODEL).
Extended Data Figure 7
Extended Data Figure 7. Arrangement of the retromer coat on membrane tubules by cryoET
3D plots that visualise the relative positions of (a) neighbouring Vps26 dimers or (b) neighbouring Vps35/Vps29 arches (see supplementary information for details). The isosurface for visualization is set at 8σ. (c, d) Flattened cylindrical projections through the boxed regions in volumes a and b respectively. Blue to red gradient colouring is proportional to pixel values. The white circle shows the position of the central Vps26 dimer or arch. (e) A close-up view at the boxed region in c with arrows indicating position and the identity of neighbours corresponding to each of six nearest-neighbour relative arrangements between Vps26 dimers. (f) Bar plot of frequency occurrence of arrangements from e for 15795 analysed Vps26 dimers. The arrangements numbered 3 and 6, where Vps26 dimers are very closely packed, are less frequent than other arrangements. Models of these relative arrangements are shown in Fig. 3b, c. (g) Density maps are shown for the local retromer structure for each of the six different relative Vps26 arrangements. Numbering corresponds to arrangements shown in Fig. 3b. Maps are radially coloured in grey, blue, green and gold for the membrane, Vps5, Vps26 and Vps35/Vps29 layers respectively. (h) Overlay of density maps for arrangements 2 and 3, and 2 and 4, showing that in some arrangements, the Vps35 arch can tilt relative to the tubule to accommodate nearby arches. The average of arrangement 2 is coloured as above, while arrangement 3 and 4 averages are coloured transparent grey.
Figure 1
Figure 1. CryoET structure of membrane-associated retromer-Vps5.
(a) Coomassie-stained SDS-PAGE of purified retromer-Vps5. (b) Section through a cryo-electron tomogram of retromer-Vps5 coated membrane tubules. a and b are representative of at least 3 independent experiments. (c) Ribbon model of retromer-Vps5 superimposed on overlapped, low-resolution electron density maps from an intermediate subtomogram alignment (Extended Data Fig. 2e). Lower-right shows three copies of the same density (one is boxed) placed at positions related by the two-fold dimeric interface formed by Vps26, illustrating how the coat can propagate around the tubule. (d) Close-up views of the retromer model fitted into the final high-resolution density maps.
Figure 2
Figure 2. Structures of interfaces within retromer-Vps5.
(a) Ribbon model of Vps26 dimer interacting with four membrane-bound Vps5 dimers. Segmented electron density for the lipid bilayer is illustrated. Top view highlights the two-fold symmetry of the Vps26-Vps5 assembly. Cross-section though the model illustrates interactions between Vps26 loops and Vps5 helices. (b) Ribbon and surface models of Vps5. Surfaces are gradient-coloured by electrostatic potential from red (negative) to blue (positive). (c) Adjacent Vps5 dimers undergo tip-to-tip interactions between BAR domain helices and lateral interactions between PX domains. (d) Overlay of ribbon model and the electron density map showing that the C-terminal α-helix of one Vps5 monomer protrudes towards the Vps26 C-terminal domain. In human Vps26, this is where Dmt1-II cargo binds in cooperation with Snx3 (Extended Data Fig. 4c, d). (e) The apex of the retromer arch viewed looking towards the membrane (left) and from the side (right). It is formed by a homodimeric interaction of Vps35 subunits (interface in blue) on the opposite face to where Vps29 is bound. Vps35 residues D694 (D620 in human Vps35) are indicated (arrows). The human mutation D620N causes Parkinson’s disease. (f) Cut-away view showing one arch leg. (g) As in f coloured by sequence conservation from red (low) to blue (high).
Figure 3
Figure 3. Organization of the retromer-Vps5 coat on membrane tubules, and the structure of retromer within the cell.
(a) A typical retromer-Vps5 coated tubule. Models of the individual elements of retromer-Vps5 have been placed at positions and orientations determined by subtomogram averaging. Left panel shows Vps5 and Vps26 layers, right panel shows the complete coat, also viewed along the tube axis of a retromer tubule. Representative models were prepared by segmentation and low-pass filtering of key features, and their respective protein structures are illustrated. (b) A model of the Vps5-Vps26 layers (corresponding to dashed box in a). Vps26 dimers dock in six relative orientations on the underlying Vps5 array, indicated by magenta arrows (Extended Data Fig. 7e). (c) A complete model of the retromer coat section shown in b. (d) Slice thorough one of 12 tomographic reconstructions of a C. reinhardtii cell in which retromer-coated membranes were identified (arrowheads). (e) Magnified views of two of 17 retromer-coated membranes in which arches can be seen. (f) Density maps filtered to 35 Å from retromer structures determined by subtomogram averaging in situ within the cell and in vitro, fitted with retromer models.
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