A scissors mechanism for stimulation of SNARE-mediated lipid mixing by cholesterol

Abstract

Neurotransmitter release at the synapse requires membrane fusion. The SNARE complex, composed of the plasma membrane t-SNAREs syntaxin 1A and SNAP-25 and the vesicle v-SNARE synaptobrevin, mediates the fusion of 2 membranes. Synaptic vesicles contain unusually high cholesterol, but the exact role of cholesterol in fusion is not known. In this study, cholesterol was found to stimulate SNARE-mediated lipid mixing of proteoliposomes by a factor of 5 at a physiological concentration. Surprisingly, however, the stimulatory effect was more pronounced when cholesterol was on the v-SNARE side than when it was on the t-SNARE side. Site-directed spin labeling and both continuous wave (CW) and pulsed EPR revealed that cholesterol induces a conformational change of the v-SNARE transmembrane domain (TMD) from an open scissors-like dimer to a parallel dimer. When the TMD was forced to form a parallel dimer by the disulfide bond, the rate was stimulated 2.3-fold even without cholesterol, supporting the relevance of the open-to-closed conformational change to the fusion activity. The open scissors-like conformation may be unfavorable for fusion and cholesterol may relieve this inhibitory factor.

Fig. 1.

Lipid-mixing assays at several different cholesterol concentrations. (A) The changes of NBD fluorescence reflecting lipid mixing. The black trace is the control run with the t-liposomes reconstituted with only Syntaxin 1A (without SNAP-25). The gray trace shows lipid mixing for both v- and t- liposomes without cholesterol. The cyan trace is the lipid-mixing assay with 20 mol % cholesterol on both sides. The red trace represents the lipid-mixing assay with 40 mol % cholesterol on both liposomes. (B) The relative initial rates of lipid mixing. The initial rates were determined by fitting the rise of the fluorescence intensity with the linear line near t = 0. (C) The inner leaflet mixing assay. The gray trace is the negative control without SNAP25. The blue trace is the fluorescence change representing inner leaflet mixing for the liposomes without cholesterol. The pink trace represents inner leaflet mixing for the liposomes with 40 mol % cholesterol. (D) The relative initial rates for inner leaflet mixing. The error bars were obtained from at least n = 4 independent measurements from 4 different preparations.

Fig. 2.

Lipid-mixing assays with asymmetric distribution of cholesterol. (A) The changes in the NBD fluorescence intensity reflecting lipid mixing. The black trace is the control run with the t-SNARE liposome reconstituted with only Syntaxin 1A (without SNAP-25). The gray trace is for the case without cholesterol. The red trace is for v-liposome with 40 mol % cholesterol but no cholesterol on t-liposome. The blue trace is vice versa. The pink trace is for 40 mol % cholesterol on both sides. (B) The relative initial rates of lipid mixing. The initial rate without cholesterol on both sides was set as 1. The error bars were obtained from at least n = 4 independent measurements from 4 different preparations.

Fig. 3.

SDSL and the EPR analysis of the spin-labeled VAMP2 TMD. (A) Room-temperature EPR spectra of spin-labeled VAMP2 mutants shown in the derivative mode. The red spectra are for the mutants reconstituted to liposomes containing 40 mol % cholesterol. The black spectra are for the proteins in the liposomes without cholesterol. (B) Low-temperature EPR spectra of VAMP2 mutants shown in the absorbance mode. For each mutant, the red spectrum is with 40 mol % cholesterol, and the black spectrum is without cholesterol. The blue spectra are the control from yeast Syntaxin-analog Sso1p N227C, which shows no spin–spin interaction (32). The single asterisk represents several dipolar broadened spectra even without cholesterol in the liposome. The double asterisk represents further broadening after the addition of 40 mol % cholesterol. (C) Plot of distances vs. the residue number for the VAMP2 TMD. In the presence of cholesterol, distances (filled circles) were measured by CW-ESR. In the absence of cholesterol, distances (<25 Å) for the residues 96–107 were measured by CW-EPR (open circles). (D) Helical wheel diagram for the VAMP2 TMD. Positions 106, 110, and 113, which showed the shorter distance with 40 mol % cholesterol, are shown in red. Positions 96, 99, and 103, which showed shorter distances even without cholesterol, are shown in white. The lipid mixtures of POPC, DOPS, and cholesterol in molar ratios of 85:15:0 and 45:15:40 were used.

Fig. 4.

Lipid-mixing assays for cross-linked VAMP2 wild-type C103 or mutant Y113C. (A) The NBD fluorescence change for cross-linked Y113C-C. (Left) The gray trace is a negative control without SNAP-25. The black trace is for non-cross-linked VAMP2 mutant Y113C without cholesterol. The red trace is for the cross-linked VAMP2 mutant Y113C-C without cholesterol. The blue and pink traces are for the non-cross-linked Y113C and cross-linked Y113C-C in the liposomes with 40 mol % cholesterol, respectively. (Right) The relative initial rates. (B) The NBD fluorescence change for cross-linked C103C-C. (Left) The cyan trace is for the lipid-mixing assay for non-cross-linked VAMP2 C103. The red trace is the cross-linked VAMP2 C103C-C. (Right) The relative initial rates.

Fig. 5.

Hypothetical models for the VAMP2 TMDs in the membrane in the absence (A) and in the presence (B) of cholesterol. In the absence of cholesterol, the dimer has an open-scissors-like conformation that may force the bilayer to have the negative curvature, which is not favorable for fusion. But in the presence of cholesterol, the dimer changes to the parallel conformation, which may not harbor the unfavorable negative curvature. The Syntaxin TMD was modeled as a tetramer based on our earlier work on yeast SNAREs (24).