Membrane fusion intermediates via directional and full assembly of the SNARE complex

Abstract

Cellular membrane fusion is thought to proceed through intermediates including docking of apposed lipid bilayers, merging of proximal leaflets to form a hemifusion diaphragm, and fusion pore opening. A membrane-bridging four-helix complex of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediates fusion. However, how assembly of the SNARE complex generates docking and other fusion intermediates is unknown. Using a cell-free reaction, we identified intermediates visually and then arrested the SNARE fusion machinery when fusion was about to begin. Partial and directional assembly of SNAREs tightly docked bilayers, but efficient fusion and an extended form of hemifusion required assembly beyond the core complex to the membrane-connecting linkers. We propose that straining of lipids at the edges of an extended docking zone initiates fusion.

Fig. 1

(A) A symmetrical toroidal stalk (top) and an expanded hemifusion diaphragm (bottom) are thought to be intermediates in the SNARE-mediated fusion pathway. (B) The ΔN complex is necessary for promoting fast N-terminal binding for trans SNARE complex formation. (C) Size distributions of small (solid) and large (dashed) liposomes reconstituted with syb determined by light scattering (see fig. S1 for characterization of the reconstitution). Inset: schematic showing liposome size dependence (for a single component system) of the elastic bending energy of a lipid in the outer monolayer (8). (D) Large liposomes (dash) exhibit a prolonged lag phase in lipid-mixing compared to small liposomes (solid) at the same SNARE density. (E) Fluorescence anisotropy of large liposomes containing syb ^28A488 (top panel) and ΔN syb 49–96^79A488 complex (bottom panel) were added to large liposomes containing their respective non-labeled SNARE binding partners, revealing that formation of trans SNARE complexes and ΔN syb displacement begin without delay. Note that anisotropy likely decreases when ΔN syb displacement has already initiated without the fragment being fully removed.

Fig. 2

Ultrastructural and biophysical identification of docking and hemifusion. (A) Negative stain EM depicting liposomes engaged in docking taken at ~3 min after mixing. Bar = 200 nm. Docked liposomes with minimal (B) and extensive (C–D) contact zones were observed by cryo-EM ~1–2 min after mixing. (E–F) Hemifused liposomes were identified by an extended diaphragm consisting of a single bilayer (arrow). Bar = 50 nm except in (F) where bar = 20 nm. (G) Discrimination of docking and fusion by FCCS-FRET. Cross-correlation between labeled liposomes (reflecting both fused and docked liposomes, red) were subtracted from changes in fluorescence lifetime (reflecting fused liposomes, black) to reveal the evolution of docked liposomes (blue). Data are mean +/− SD (N ≥ 5). (H) Total (black) and inner leaflet (red) lipid-mixing measured and compared to the expected inner leaflet lipid-mixing (cross-shaded region). Hemifusion begins to form after ~3–4 min and accumulates thereafter. Bars represent 95 % confidence intervals (N ≥ 3).

Fig. 3

Disruption of the C-terminal +8 layer of the SNARE complex arrests a tightly docked intermediate. (A) Comparative lipid-mixing of wild-type syb (black) and syb Δ84 (red) according to the depicted liposome size combinations (location of ΔN complex is indicated in light blue), showing curvature affects the ability of syb Δ84 to mediate fusion. Traces were normalized to wild-type syb which was set arbitrarily to 1. (B) FCCS-FRET analysis of large syb Δ84 liposomes showing accumulation of docking. Data are mean +/− SD (N ≥ 5). (C) Examples of cryo-EM images of syb Δ84 and ΔN complex large liposomes depicting the arrest at the tightly docked state. The edges of an extended docking zone results in straining of lipids (arrow). Bar = 20 nm. (D) Counting of docking of syb Δ84 and ΔN complex liposomes observed by cryo-EM after 1 h, confirming the vast majority of liposomes were arrested in the tightly docked state. Control was performed in the presence of excess soluble syb 1–96 showing that docking was SNARE-dependent.

Fig. 4

Full assembly of the core SNARE complex restores hemifusion and the linkers are needed for efficiently completing fusion. (A) Comparative lipid-mixing of linker deletion mutants depicting a decrease in fusion in large liposomes. (B) Inner leaflet lipid-mixing as a proportion of expected assuming full-fusion conditions for wild-type syb and linker deletion mutants. Values were taken 1 h after mixing and bars represent 95 % confidence intervals (N ≥ 3). (C) Ribbon structure (21) of the fully assembled SNARE complex showing the interacting layers (black lines, +8 layer indicated) of the four-helix bundle, the linker and TMD and the proposed regions which give rise to docking, extended hemifusion and fusion. Dashed lines indicate borders of regions that are based on other studies (22, 23) or on biochemical characterization. Inset: deleted amino acids used for mutation analysis.