Curvature induction and membrane remodeling by FAM134B reticulon homology domain assist selective ER-phagy

Abstract

FAM134B/RETREG1 is a selective ER-phagy receptor that regulates the size and shape of the endoplasmic reticulum. The structure of its reticulon-homology domain (RHD), an element shared with other ER-shaping proteins, and the mechanism of membrane shaping remain poorly understood. Using molecular modeling and molecular dynamics (MD) simulations, we assemble a structural model for the RHD of FAM134B. Through MD simulations of FAM134B in flat and curved membranes, we relate the dynamic RHD structure with its two wedge-shaped transmembrane helical hairpins and two amphipathic helices to FAM134B functions in membrane-curvature induction and curvature-mediated protein sorting. FAM134B clustering, as expected to occur in autophagic puncta, amplifies the membrane-shaping effects. Electron microscopy of in vitro liposome remodeling experiments support the membrane remodeling functions of the different RHD structural elements. Disruption of the RHD structure affects selective autophagy flux and leads to disease states.

Fig. 1

Sequence and topology of FAM134B. a Schematic of the full length FAM134B sequence. The RHD consists of two transmembrane segments (green, TM12 and TM34) separated by a 60-residue linker, and two additional terminal segments. The C-terminal fragment of the RHD and the linker-helix (yellow) form conserved amphipathic helices. b Topology of FAM134B-RHD (80–260). Charged residues (K/R blue and D/E red), TM segments (green) and amphipathic helices (yellow) are highlighted. Genetic variant Q145X and proteolytic cleavage product R142X result in truncated proteins (red dotted line) disrupting the RHD. The N-terminal and C-terminal disordered regions (not modeled, gray) flank the RHD on the cytosolic face of the ER membrane

Fig. 2

3D structural model of FAM134B-RHD from MD simulations. a, b Transmembrane fragments fold into helical hairpins (red and blue helices in (a) TM12 and (b) TM34. Flanking charged and polar residues and luminal loop residues anchor the two hairpins within the ER membrane (labeled side chains). c, d Linker and C-terminal fragments form amphipathic helices (c) AH_L and (d) AH_C (yellow cartoon). Polar (colored labels) and apolar residues (yellow labels) on opposite sides position the helices at the water–bilayer interface. e Overlapping, individually refined fragment structures were used to assemble the FAM134B-RHD (80–260) structural model. The model was first equilibrated using coarse-grained simulations and then refined with all-atom MD simulations. f Time-averaged local membrane profile (gray mesh; top, side, and 3D views) around FAM134B-RHD (colored) computed from all-atom MD simulations displays perturbations of the bilayer structure

Fig. 3

Bicelle-to-vesicle transitions. a, b Snapshots showing curvature of bicelles containing DMPC (gray) and short chain DHPC lipids (red) (a) with KALP₁₅ peptide (blue) and (b) FAM134B-RHD (green). c KALP₁₅-containing bicelles remain flat with low curvature (|H| = ±0.005 nm⁻¹), rarely displaying vesiculation events (5/96) within 96 simulations of 1000 ns each. d FAM134B-RHD actively curves the bicelle to form vesicles in repeated runs (92/95). FAM134B-RHD induces strong positive curvature along the cytoplasmic leaflet resulting in positively curved vesicles (H = +0.16 nm⁻¹). c, d Curvature time-traces (blue/green, smoothed running averages over 11 ns widows) from individual replicates quantify the bicelle shape transformation process during simulations. **Denote and n.s. denote the one-tailed probability in binomial tests for bias in number of vesiculation events with positive and negative bicelle curvatures

Fig. 4

Curvature sensing by FAM134B-RHD. a Cut through the simulation box along the xz plane (inset top view) showing the buckled lipid bilayer (orange phosphate beads), with excess area (≈17 nm²) under edge compression. Diffusion of curvature-inducing proteins such as FAM134B-RHD (green) in the buckled membrane enables curvature sampling and estimation of intrinsic curvature preferences (see the section “Methods”). We tracked the position of proteins (x, y) along the buckle, and quantified the curvature preference (principal, mean and Gaussian; see Supplementary Fig. 15). b Histograms of mean curvature, H(x, y), sampled by FAM134B-RHD (green) in coarse-grained simulations (1 ns intervals for 20 μs) indicate a preference for highly curved regions of the buckle. By contrast, the KALP₁₅ peptide (blue) samples regions with lower curvature along the buckle. The local mean curvature field of the empty buckled membrane (red) is obtained by random sampling of points in the xy plane (see Supplementary Fig. 16)

Fig. 5

RHD unique topology drives curvature and clustering in membranes. a Snapshot from coarse-grained simulation. The FAM134B-RHD forms a wedge-shaped protein inclusion (gray shade) in the membrane (orange PO4 beads). Local bilayer thinning by short hairpins (TM12 and TM34; green) promotes inter-hairpin interactions (blue) at the luminal leaflet (see Supplementary Fig. 19). AH_L (orange/yellow sticks) separates the two hairpins on the cytosolic leaflet and, along with AH_C (yellow sticks) enhances curvature. b Simulation snapshots showing local clustering of multiple FAM134B-RHD molecules (red) on model ER membrane under periodic boundary conditions (see Supplementary Fig. 21). c Cross section of a closed tubular structure (k₁ ≈ 0.16 nm⁻¹; k₂ ≈ 0 nm⁻¹; gray with orange PO4 beads) containing 10 FAM134B-RHD molecules simulated in explicit solvent (~3.6 × 10⁶ water beads; blue). Deformed tubule structure (below) after ≈7 μs showing organization of RHDs into three local clusters. d Zoom-in on the boxed cluster containing three RHDs (see Supplementary Fig. 23 for other clusters). Side views (left) of the RHD cluster shaped as an inverted pyramid display locally curved tubule surface along principal axes, k₂ and k₁. Top view (right) showing the organization of AHs at the base of the pyramid. Two RHDs (blue and gray) align their AHs perpendicular to the tube axis, while the AHs of the third RHD (yellow) are parallel to the tube axis

Fig. 6

RHD structure determines in vitro membrane binding and liposome remodeling activity. a Liposome co-flotation assay to evaluate membrane-binding properties of FAM134B-RHD and various deletion mutants (see the section “Methods”). Purified protein samples were incubated with liposomes for 2 h at 37 °C and subjected to flotation on a sucrose cushion (top to bottom, 1–8) followed by SDS–PAGE and western blotting with anti-GST antibody. b–i Representative nsTEM micrographs of remodeled proteoliposomes (scale bars, 200 nm). Empty liposomes (b) were remodeled by incubation after addition of purified (c) GST, (d) wild-type RHD, (e) ΔAH_L + AH_C, (f) ΔTM12, (g) ΔTM34, (h) ΔTM12 + TM34, and (i) RHD_143–260 for 18 h at 22 °C. Insets (red squares); magnified micrographs showing examples of representative proteoliposomes with diameters measured (dotted lines) using ImageJ. j Violin plots show the measured proteoliposome size-distributions (n = 300 each) from nsTEM images. Violins shows a central boxplot (median with interquartile range, black lines) along with mirrored histograms on either sides (colored)

Fig. 7

TM hairpins of FAM134B are required for ER fragmentation. a Immunofluorescence of HA and endogenous calnexin in U2OS cells transiently overexpressing HA-tagged FAM134B (left to right): wild-type (WT), LIR mutant (LIR mut), single hairpin deletions (ΔTM12 and ΔTM34), and the double hairpin deletion (ΔTM12 + TM34). Scale bars 10 μm. b Quantification of U2OS cells with fragmented ER (≥5 ER fragments per cell) after transient over-expression of wild-type and mutant forms of FAM134B. Error bars indicate s.d. from triplicates (red filled circles) and *Denotes p < 0.05 in two-sample Student’s t-tests. c Immunofluorescence of endogenous calnexin in un-transfected U2OS cells. Scale bar 10 μm

Fig. 8

Role of FAM134B-induced membrane curvature in ER-phagy. FAM134B induces high curvature in the ER membrane (yellow/orange). The high intrinsic curvature preference of the FAM134B-RHD (dark blue helices) enables its partitioning and clustering specifically to perinuclear ER. The presence of N-terminal and C-terminal disordered fragments (black lines) enhances local curvature (yellow gradient) by providing additional steric forces to bend the ER membrane. Using the C-terminal LIR (pink box), FAM134B also forms a physical bridge between the ER membrane (orange) and phagophore membrane (gray)-associated LC3-PE (yellow/green). This interaction provides additional forces required for scission and fragmentation of ER membranes. High local membrane curvature induced by FAM134B-RHD thus lowers the barrier for membrane budding and subsequent pinch-off in FAM134B-enriched ER