Clustering of syntaxin-1A in model membranes is modulated by phosphatidylinositol 4,5-bisphosphate and cholesterol

Abstract

Syntaxin-1A is part of the SNARE complex that forms in membrane fusion in neuronal exocytosis of synaptic vesicles. Together with SNAP-25 the single-span transmembrane protein syntaxin-1A forms the receptor complex on the plasma membrane of neuroendocrine cells. Previous studies have shown that syntaxin-1A occurs in clusters that are different from lipid rafts in neuroendocrine plasma membranes. However, the interactions that promote these clusters have been largely unexplored. Here, we have reconstituted syntaxin-1A into lipid model membranes, and we show that syntaxin cluster formation depends on cholesterol in a lipid system that lacks sphingomyelin and therefore does not form liquid-ordered phases that are commonly believed to represent lipid rafts in cell membranes. Rather, the cholesterol-induced clustering of syntaxin is found to be reversed by as little as 1-5 mol % of the regulatory lipid phosphatidylinositol 4,5-bisphosphate (PI-4,5-P(2)), and PI-4,5-P(2) is shown to bind electrostatically to syntaxin, presumably mediated by the highly positively charged juxtamembrane domain of syntaxin. Possible implications of these results to the regulation of SNARE-mediated membrane fusion are discussed.

Figure 1

Cholesterol promotes the formation of syntaxin clusters in lipid model membranes as measured by self-quenching of fluorescently labeled syntaxin-1A. A. Cartoon model of cholesterol-induced cluster formation of syntaxin. Arrows represent observed fluorescence. B. Representative fluorescence emission spectra of Alexa647-labeled syntaxin-1A reconstituted in POPC liposomes with increasing concentrations of cholesterol. The protein-to-lipid ratio is 1:1000. C. Scaled fluorescence intensities averaged from at least three experiments like the one shown in panel B as a function of cholesterol concentration. D. Absorbance (at 650 nm) and fluorescence emission (at 671 nm) intensities of Alexa647-labeled syntaxin-1A in the presence and absence of 20 mol % cholesterol, indicating that the protein is incorporated at the same levels in POPC bilayers with and without cholesterol. All values are averages of at least three samples. Absorbance error bars represent three standard deviations for ease of observation, and emission errors are displayed as one standard deviation. Similar data were obtained at other cholesterol concentrations.

Figure 2

Acidic lipids disperse cholesterol-induced clusters of syntaxin in lipid model membranes as measured by self-quenching of fluorescently labeled syntaxin-1A. A. Cartoon model of syntaxin cluster dispersion by acidic lipids (red headgroups marked with “−“ charges). B. Scaled fluorescence intensities of Alexa647-labeled syntaxin-1A in lipid bilayers of lipid different compositions. The fluorescence intensity in POPC:CHOL (4:1) is self-quenched as in Figure 1. Various additions of acidic lipids relieve this self-quenching. All intensities are scaled to the syntaxin in POPC only fluorescence. The means and standard deviations from at least three independent reconstitutions for each condition are shown. C. Scaled fluorescence intensities of Alexa647-labeled syntaxin-1A as a function of PI 4,5 P₂ concentration in POPC lipid bilayers containing 20% cholesterol. The means and standard deviations from at least three independent reconstitutions for each condition are shown.

Figure 3

Cholesterol-induced clustering and acidic lipid-induced cluster dispersion of syntaxin in lipid model membranes as measured by FRET between two fluorescently labeled syntaxins. A. Cartoon model of the protein-protein FRET experiments in lipid bilayers. B. Excitation and emission fluorescence spectra of the labeled proteins and lipids reconstituted into liposomes that were used in this work: Bodipy-TMR-PIP2-C16 (blue), Alexa546-labeled syntaxin-1A (green), and Alexa647-labeled syntaxin-1A (red). C. Mean FRET efficiencies in reconstituted lipid bilayers with Alexa546-labeled syntaxin and Alexa647-labeled syntaxin. Each protein is incorporated at a protein-to-lipid concentration of 1:2000. The presence of cholesterol significantly increases the energy transfer efficiency and this effect is reversed by acidic lipids. The means and standard deviations from at least three independent reconstitutions for each condition are shown.

Figure 4

PI-4,5-P₂ associates with syntaxin in lipid bilayers containing cholesterol as measured by lipid-to-protein FRET. A. Cartoon model of the lipid-protein FRET experiments in lipid bilayers. B. Mean observed FRET efficiencies from BODIPY-TMR-PIP2-C16 to Alexa647-labeled syntaxin-1A in POPC bilayers with and without cholesterol and at different salt concentrations. The means and standard deviations from at least three independent reconstitutions for each condition are shown. C. Bodipy-TMR-PIP2-C16 fluorescence intensities in protein-free liposomes of different compositions and scaled to the fluorescence in POPC showing that the fluorescent probes are approximately equally fluorescent in all lipid backgrounds, except in liposomes containing cholesterol and PI-4,5-P₂, where the fluorescence intensity is about 30 % greater.

Figure 5

“Icebreaker” model of PI-4,5-P₂ action on cholesterol induced syntaxin clustering and its proposed role in synaptic vesicle fusion. Synaptic vesicles are proposed to dock to nanoclusters of t-SNAREs (syntaxin and SNAP 25). Upregulation of the PI-4,5-P₂ concentration in the plasma membrane causes increased binding of this lipid to the polybasic juxtamembrane domain of syntaxin and dissociates fusogenic oligomers of t-SNAREs with docked vesicles from the nanoclusters. Lipids with green headgroups designate triple negatively charged PI-4,5-P₂. The + signs on the syntaxins symbolize their polybasic juxtamembrane region. Cholesterol (polycyclic lipid structures) competes with syntaxin for solvation by bulk lipid.