Neuronal SNAREs do not trigger fusion between synthetic membranes but do promote PEG-mediated membrane fusion

Abstract

At low surface concentrations that permit formation of impermeable membranes, neuronal soluble N-ethyl maleimide sensitive factor attachment protein receptor (SNARE) proteins form a stable, parallel, trans complex when vesicles are brought into contact by a low concentration of poly(ethylene glycol) (PEG). Surprisingly, formation of a stable SNARE complex does not trigger fusion under these conditions. However, neuronal SNAREs do promote fusion at low protein/lipid ratios when triggered by higher concentrations of PEG. Promotion of PEG-triggered fusion required phosphatidylserine and depended only on the surface concentration of SNAREs and not on the formation of a trans SNARE complex. These results were obtained at protein surface concentrations reported for synaptobrevin in synaptic vesicles and with an optimally fusogenic lipid composition. At a much higher protein/lipid ratio, vesicles joined by SNARE complex slowly mixed lipids at 37 degrees C in the absence of PEG, in agreement with earlier reports. However, vesicles containing syntaxin at a high protein/lipid ratio (>or=1:250) lost membrane integrity. We conclude that the neuronal SNARE complex promotes fusion by joining membranes and that the individual proteins syntaxin and synaptobrevin disrupt membranes so as to favor formation of a stalk complex and to promote conversion of the stalk to a fusion pore. These effects are similar to the effects of viral fusion peptides and transmembrane domains, but they are not sufficient by themselves to produce fusion in our in vitro system at surface concentrations documented to occur in synaptic vesicles. Thus, it is likely that proteins or factors other than the SNARE complex must trigger fusion in vivo.

FIGURE 1

Cartoon summarizing the procedures leading to intervesicle SNARE complexes. Details are mentioned in Materials and Methods and Results. Solid and open stars indicate the positions of fluorescent probes fluorescein and tetramethylhodamine, respectively. The open circle indicates the label position of fluorescein in syntaxin (Fa-SX) used to detect antiparallel orientation of SX and SB.

FIGURE 2

Demonstration of directed SX reconstitution into membrane vesicles. (A) Fluorescence (○) and turbidity (□) of F-SX vesicles, fluorescence of rhodamine-labeled SUV (▵), and fluorescence of detergent-solubilized F-SX (⋄) versus the fraction number collected from a Sepharose CL4B column. The inset shows a plot of fluorescence intensity of F-SX vesicles versus their turbidity. (B) SDS-PAGE of SX vesicles digested with CT before and after vesicle disruption by Triton X-100 (TX-100).

FIGURE 3

Time courses of SNARE complex assembly followed by light scattering and by FRET from fluorescein-labeled F-SX vesicles to tetramethylrhodamine-labeled R-SB vesicles. (A) Mean diameters of SN25:SX vesicles plus SB vesicles (•), SX vesicles plus SB vesicles (▪), protein-free control vesicles (○), and control vesicles plus SN25 (□), SN25:Fa-SX vesicles plus R-SB vesicles (▴) as a function of incubation time with 3 wt % PEG. Before QELS measurements, vesicle samples were diluted to <0.1 wt % PEG (aggregation limit 1 wt %). (B) FRET (F_D/F_DA) for the same vesicle combinations and conditions described for frame A, with symbols as in frame A, with F-SX and R-SB replacing SX and SB. F_D/F_DA for SN25:F-SX vesicles plus R-SB vesicles when incubated without PEG is shown as solid diamonds. FRET from F-SX vesicles to R-SN25 (⋄) shows the assembly of the SX/SN25 binary complex. FRET from SN25:Fa-SX vesicles to R-SB vesicles (▴) tests for assembly of antiparallel SNARE complex. (C) Stability of assembled SNARE complex (as detected by FRET efficiency) as a function of time after diluting PEG with symbols as in frame A and B except that the inverted open triangles show the disassembly of complex assembled from SX vesicles plus SB vesicles and SN25 without the preassembly of SX vesicles:SN25 complex.

FIGURE 4

Estimation of complex assembly by FRET. (A) FRET (F_D/F_DA) from F-SX to R-SB (both β-OG solubilized) is plotted versus the concentration of R-SB added to a solution of F-SX:SN25 binary complex, with dye labels positioned to detect the parallel configuration (•) and the antiparallel configuration (▴) between SX and SB. A mixture of 5 μM F-SX and 10 μM SN25 was incubated for 30 min to produce F-SX:SN25 complex before adding varying amounts of R-SB and incubating for 24 h to form the full ternary complex. A small amount of this incubated mixture was diluted to 0.1 μM in F-SX for fluorescence measurements. The inset shows SDS-PAGE analysis without boiling samples of SNARE complex assembled from solubilized full-length SX (7.5 μM), SN25 (15 μM), and truncated SB (residues 1–96) after 24 h of incubation. Concentrations of SB were 2 and 5 times that of SX. Band intensities were digitized to calculate the percentage of SX in SNARE complex, and these values are shown as solid squares for comparison to the FRET values from labeled SNARE complex.

FIGURE 5

Effects of SNARE complexes on fusion. Time courses (at 23°C) of PEG- (6 wt %) induced (A) lipid mixing, (B) contents mixing, and (C) contents leakage of DOPC/DOPE/SM/CH/DOPS SUVs for protein-free control vesicles (black), SX-V:SB-V (red), control vesicles plus SN25 (blue), and SN25:SX-V:SB-V (green). Solid black lines through these time courses show the best fits of these time courses to single-exponential curves (see Materials and Methods). The invariant green dotted lines show time courses observed for SN25:SX-V:SB-V complexes at 23°C when no PEG was added to vesicles joined by stable SNARE complexes (SN25:SX-V:SB-V). The inset in frame A shows the time courses of lipid mixing for the same vesicle combinations described in frame A at 37°C when no PEG was present, conditions for which content mixing was not observed.

FIGURE 6

Effects of increased surface content of SNARE proteins. Time courses at 23°C of PEG- (6 wt %) mediated (A) lipid mixing, (B) contents mixing, and (C) contents leakage involving DOPC/DOPE/SM/CH/DOPS SUVs containing SX and SB at 1:980 and 1:420 protein/lipid ratios, respectively. Data are presented for protein-free control vesicles (black lines), SX-V:SB-V complexes (red), control vesicles plus SN25 (blue lines), and SN25:SX-V:SB-V complexes (green lines). Solid black lines on top of these time courses are the best fit obtained when these time courses were fit to a double-exponential expression. The dashed black lines in panels A and B show single-exponential fits for the time courses of SX-V:SB-V complexes (red) and SN25:SX-V:SB-V complexes (green). The invariant dotted green lines show time courses taken for SN25:SX-V:SB-V complexes at 23°C in the absence of PEG. The inset in frame A shows lipid-mixing kinetics for the same vesicle combinations described in frame A at 37°C in the absence of PEG, conditions for which content mixing was not observed.

FIGURE 7

SNARE-induced lipid mixing and effect of SNARE reconstitution on loss of trapped contents. (A) Percentage of trapped contents of SX vesicles (gray bars) and SB vesicles (black bars) after single, double, triple, and quadruple reconstitutions. Percentage of trapped content was obtained from the difference in the intensities of Tb and DPA coencapsulated in vesicles before and after lysis using C₁₂E₈ detergent. These values were compared to those obtained for control vesicles at the respective reconstitution step to calculate percentage of trapped contents. (B) Lipid-mixing time courses of quadruple-reconstituted control vesicles (dashed line) and vesicles joined by stable SNARE complex (gray circles) incubated at 37°C in the absence of PEG. Samples incorporated SX and SB at P/L ratios of ∼1:250 and 1:120, respectively. The inset shows time courses of lipid mixing at 23°C between control vesicles, vesicles joined by stable SNARE complex, and SB vesicles plus SB vesicles (all indicated by arrows), as triggered by 6% PEG. Gray dots show the data for SNARE-linked vesicles obtained in the absence of PEG, for reference. Solid black lines represent best fits of single (control in the inset and SNARE vesicles absent PEG at 37°C) or double exponentials to the data.

FIGURE 8

Effect of PS on SNARE effects on fusion. Time courses of (A) lipid mixing, (B) contents mixing, and (C) contents leakage involving DOPC/DOPE/SM/CH SUVs at 3 wt % PEG for protein-free control vesicles (black), SX vesicles plus SB vesicles (red), control vesicles plus SN25 (blue), and SN25/SX vesicles plus SB vesicles (green) with best fit shown as solid black lines. The green dotted lines show the time courses when no PEG was added to SN25/SX vesicles plus SB vesicles.

FIGURE 9

Cartoon summarizing the roles of SNAREs on PEG-mediated fusion of SB- and SX-containing SUVs. SX promotes stalk intermediate formation, fast pore formation in the stalk state, and evolution of the stalk intermediate to a fusion pore.