Molecular mechanism of cholesterol- and polyphosphoinositide-mediated syntaxin clustering

Abstract

The neuronal acceptor SNARE complex that functions as the receptor for synaptic vesicle docking and fusion at the presynaptic membrane is composed of the single-span transmembrane protein syntaxin-1A and the palmitoylated soluble protein SNAP-25. Previously, we explored interactions that promote the formation of syntaxin-1A clusters in membranes. Cholesterol activates clustering in native and model membranes, and its depletion in neuroendocrine cells results in a homogeneous distribution of the protein. However, as little as 1 mol % phosphatidylinositol 4,5-bisphosphate (PI-4,5-P(2)) or 20 mol % phosphatidylserine was found to disperse syntaxin-1A clusters [Murray, D. H., and Tamm, L. K. (2009) Biochemistry 48, 4617-4625]. Strong evidence suggests that syntaxin-1A and its synaptic vesicle cognate synaptobrevin both interact directly with PI-4,5-P(2) and that this interaction activates fusion. However, the molecular details of this interaction and its relationship to the partial dispersion of syntaxin-1A clusters remain largely unexplored. Hence, we mutated the polybasic juxtamembrane motif of syntaxin-1A and found several residues that partially or fully abrogate the electrostatic interaction with PI-4,5-P(2). We further show that even in the presence of physiological concentrations of phosphatidylserine, the PI-4,5-P(2)-syntaxin interaction is sufficiently strong to disrupt syntaxin-1A clustering. The stereochemistry of PI-4,5-P(2) is not critical for this interaction as other polyphosphoinositides have similar effects. Forming an acceptor SNARE complex between syntaxin-1A and SNAP-25 weakens but does not abrogate cholesterol/PI-4,5-P(2)-controlled cluster formation. Potential consequences of these interactions with respect to synaptic vesicle fusion are discussed.

Figure 1

Sequence alignment of the juxtamembrane domain regions of human syntaxins. The alignment (generated in BioEdit () using a database of SNAREs ()) reveals a conserved polybasic region that is most pronounced in family members that are targeted to the plasma membrane. The final layers of the four-helical bundle making up the SNARE core complex and the entire transmembrane domains concluding with the C-termini are also shown flanking the juxtamembrane regions.

Figure 2

Cholesterol-dependent clustering of syntaxin-1A in lipid model membranes is relieved by the anionic lipids POPS and PI-4,5-P₂ as measured by self-quenching of Alexa 647-labeled syntaxin. A. Cartoon model of syntaxin clustering and the resulting self-quenching that is reduced in the presence of anionic lipids. B. Scaled fluorescence intensities of labeled syntaxin reconstituted into liposomes of different lipid composition at protein-to-lipid ratios of 1:1000. The data represent averages of four or more independent experiments with error bars indicating single standard deviations. Paired bars are statistically significantly different at confidence levels of p<0.05 (*) and p<0.01(**).

Figure 3

Dispersion of syntaxin-1A clusters by different polyphosphoinositides in lipid model membranes is not specific for the phosphate position on the lipid headgroup. Clustering was measured by self-quenching of labeled syntaxin in POPC:Chol (4:1) bilayers containing 1 mol% of the indicated phosphoinositide. The fluorescence is scaled (fraction recovery) to control samples with syntaxin in POPC and POPC:Chol bilayers without phosphoinositides that were measured in each experimental series to establish 1 and 0 fraction recovery, respectively. Data represent averages of four or more independent experiments with error bars indicating single standard deviations.

Figure 4

Role of individual basic residues of polybasic juxtamembrane motif in dispersion of cholesterol-induced syntaxin-1A clusters by binding of PI-4,5-P₂. For each mutant, fluorescence self-quenching experiments were performed in POPC, POPC:Chol (3:2), and POPC:Chol (3:2) plus 3 mol% PI-4,5-P₂. A. Experiments with wild-type, and RRAA, AAKK, AAAA mutants. B. Experiments with single basic residue mutants RAAA, ARAA, AAKA, and AAAK. The two lysines seem mostly responsible for the cluster dispersion interaction with PI-4,5-P₂ although additional cumulative effects of all basic residues in this region are also observed. Protein-to lipid ratios were 1:1000 and measurements represent averages of four or more experiments with error bars indicating single standard deviations.

Figure 5

SNAP-25 partially disrupts clustering of syntaxin-1A in cholesterol-containing lipid model membranes. Disruption is only observed when SNAP-25 is co-reconstituted as a complex with syntaxin. Titration of SNAP-25 up to 10-fold excess over syntaxin to preformed syntaxin clusters in cholesterol-containing bilayers has no effect, i.e. is unable to disrupt these clusters. Mean scaled fluorescence intensities of labeled syntaxin at protein:lipid ratios of 1:1000 are shown for each lipid composition and SNAP-25 condition.

Figure 6

The clustering of syntaxin-1A in cholesterol-containing lipid model membranes decreases with increasing temperature as revealed by relief of self-quenching of fluorescently labeled syntaxin. A. Fluorescence intensities of 1 μM Alexa 647-labeled syntaxin in 2% v/v Triton X-100 as a function of temperature. B. Scaled fluorescence intensities of labeled syntaxin reconstituted into either POPC (closed symbols) or POPC:Chol (open symbols) bilayers at protein-to-lipid ratios of 1:1000. Three representative experiments are shown for each lipid composition. The data are normalized with the values in POPC bilayers at 20 °C set to one and corrected with the fluorophor temperature coefficient determined from graph A as described in the text. C. Mean scaled fluorescence intensities of labeled syntaxin in different environments from three or more experiments as indicated. 2% v/v Triton X-100 was added to the samples labeled with “det”.