Structural insights into membrane remodeling by SNX1

Abstract

The sorting nexin (SNX) family of proteins deform the membrane to generate transport carriers in endosomal pathways. Here, we elucidate how a prototypic member, SNX1, acts in this process. Performing cryoelectron microscopy, we find that SNX1 assembles into a protein lattice that consists of helical rows of SNX1 dimers wrapped around tubular membranes in a crosslinked fashion. We also visualize the details of this structure, which provides a molecular understanding of how various parts of SNX1 contribute to its ability to deform the membrane. Moreover, we have compared the SNX1 structure with a previously elucidated structure of an endosomal coat complex formed by retromer coupled to a SNX, which reveals how the molecular organization of the SNX in this coat complex is affected by retromer. The comparison also suggests insight into intermediary stages of assembly that results in the formation of the retromer-SNX coat complex on the membrane.

Keywords: SNX1; coat complex; cryoelectron microscopy; helical reconstruction; membrane deformation.

Fig. 1.

SNX1 is sufficient for membrane binding and tabulation. (A) Domain organization of SNX1 (NP_062701.2). PX, Phox homology domain; BAR domain. (B) Binding of SNX1 to liposomes of varying sizes (as indicated) is assessed by coprecipitation assay. Supernatant, S; Pellet, P. (C) Negative-stain EM visualizing liposomes incubated either with (first row) or without (second row) SNX1 (scale bar, 200 nm). The diameters of the liposomes are also indicated. (D) Statistical histogram of the diameters of tubules generated by SNX1 is plotted as a percentage of all tubules; n = 41 for 50 nm liposomes, n = 62 for 100 nm liposomes, n = 60 for 200 nm liposomes, n = 61 for 400 nm liposomes, and n = 70 for 1,000 nm.

Fig. 2.

Cryo-EM and three-dimensional reconstructions of SNX1 coated tubules. (A) Raw cryo-EM micrographs of tubules coated with SNX1. A regular tube is boxed in red (scale bar, 50 nm). (B) Helical diffraction patterns of tubules exemplifying class I. (C) Cryo-EM map of a class I tubule, with the side view on the top and cross-section view on the bottom, with a lower threshold in cross-section view to show the inner membrane more clearly. The map is colored according to the cylinder radius from red to blue. (D) The structural models of SNX1 in cartoon representation are fitted into the map. The PX domain and BAR domain are colored in gold and blue, respectively. (E) Structural model of the SNX1 helical assembly for the class I tubules. Interface I and II are indicated by black circles, with zoom-in views also shown.

Fig. 3.

An amphipathic helix on the membrane interaction surface of SNX1. (A) Ribbon model of the SNX1 dimer interacting with the membrane. The colors of the PX and BAR domains are the same as shown in Fig. 2E. The amphipathic helix is highlighted in green. Dashed black lines denote cross sections that are rotated by 90° and shown in B. (B) Corresponding cross sections of the tube along the dashed black lines 1 and 2 in A; shown is the fitness between the structural model and the map with high threshold. (C) The cross section rotated by 30° from that shown in B, which provides the view from the membrane. (D) Ribbon model of the amphipathic helix, with charged surface on one side and hydrophobic surface on the other side and key residues labeled. (E) Electrostatic surface representation on the concave surface of SNX1, with positive charges colored blue and negative charges colored red. Positively charged patches and other areas implicated in membrane binding and tubulation are labeled. (F) A Ribbon model of SNX1 with the location of charged patches in the BAR domain shown in magenta, the amphipathic helix shown in green, and the predicted PIP3 binding site of the PX domain shown in cyan. A hypothetical membrane is depicted based on the diameter (∼40 nm⁻¹) of tubules observed in EM maps.

Fig. 4.

Functional mutagenesis assessment of membrane binding and tabulation. (A) Binding of mutant forms of SNX1 (as indicated) to liposomes is assessed by the coprecipitation assay. Supernatant, S; Pellet, P. (B) Quantitation from three independent experiments is shown. All error bars represent SD from three independent experiments. Statistical analysis was performed comparing the wild-type and different mutants, with **P < 0.001 and *P < 0.01. (C) Negative-stain EM visualizing tubules induced by mutant forms of SNX1 (as indicated) (scale bar, 200 nm). (D) Confocal image of a HeLa cell expressing WT or mutant forms of GFP-tagged SNX1. Inset highlights endosomal tubulation seen in cells expressing WT, but not mutant, forms. (Scale bar, 10 μm.) (E) Quantitation of the above confocal analysis from three independent experiments. Error bars represent SD from three independent experiments.

Fig. 5.

Structural comparisons between the SNX1 structure solved in this study versus those previously solved that contain SNX1 or SNX1-like protein. (A) Structural comparison of a SNX1 dimer solved in the context of the membrane versus the BAR domain of SNX1 solved previously in solution (PDB: 4FZS). The membrane structure of the SNX1 dimer is colored in blue while the solution structure of the BAR-domain dimer is colored in green. (B) Structural comparison of the SNX1 dimer solved in the current study versus the Vps5 dimer from Chaetomium thermophilum (PDB: 6H7W). The SNX1 dimer is colored in blue and the Vps5 dimer is colored in pink. (C) Structural comparison of the SNX1 assembly solved in the current study versus the Vps5 assembly solved previously in the context of a retromer-Vps5 assembly. Four dimers are shown in different colors which are labeled as N, N+1, N+6, and N+7. The distances between the centroids of adjacent dimers in the same helical row and also between the PX domains in different helical rows are labeled and indicated by black lines. Protein–protein interactions are highlighted by dashed red circles. (D) A cartoon representation of conformational changes in the SNX assembly upon the further binding by retromer.