Mechanism of arachidonic acid action on syntaxin-Munc18

Abstract

Syntaxin and Munc18 are, in tandem, essential for exocytosis in all eukaryotes. Recently, it was shown that Munc18 inhibition of neuronal syntaxin 1 can be overcome by arachidonic acid, indicating that this common second messenger acts to disrupt the syntaxin-Munc18 interaction. Here, we show that arachidonic acid can stimulate syntaxin 1 alone, indicating that it is syntaxin 1 that undergoes a structural change in the syntaxin 1-Munc18 complex. Arachidonic acid is incapable of dissociating Munc18 from syntaxin 1 and, crucially, Munc18 remains associated with syntaxin 1 after arachidonic-acid-induced syntaxin 1 binding to synaptosomal-associated protein 25 kDa (SNAP25). We also show that the same principle operates in the case of the ubiquitous syntaxin 3 isoform, highlighting the conserved nature of the mechanism of arachidonic acid action. Neuronal soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs) can be isolated from brain membranes in a complex with endogenous Munc18, consistent with a proposed function of Munc18 in vesicle docking and fusion.

Figure 1

Action of arachidonic acid on plasma membrane syntaxins assessed by Coomassie-stained SDS–polyacrylamide gel electrophoresis. (A) Degree of SNARE assembly varies between syntaxins (Stx) 1 to 4 as a function of arachidonic acid (AA, 100 μM). SNAP25B and synaptobrevin 2 (Syb) were used. Boiling disrupts SNARE complexes, thus showing input protein quantities. (B) The graph shows the kinetics of SNARE assembly by syntaxin 1. A representative gel is shown with AA induced increases in the SNARE complex. AA, 100 μM. (C) Syntaxin 1 pre-treated with 100 μM AA (and then purified by gel filtration, not shown) still requires AA addition for efficient SNARE assembly. (D) Specificity of AA action on syntaxin 1. The left-hand gel shows SNARE reactions in the presence of 100 μM saturated palmitic (C16:0), stearic (C18:0), arachidic (C20:0) acids, brain lysophosphatidylcholine (LysoPC) and unsaturated AA (20:4). The right-hand gel shows SNARE assembly in the presence of C20 fatty acids with increasing unsaturation. EDA, eicosadienoic acid (C20:2); ETA, eicosatrienoic acid (C20:3). (E) Titration of AA, dodecanoic (C12:0), palmitic and arachidic acids in the SNARE assembly assay. EC₅₀ for syntaxin 1 activation by AA is about 60 μM. SNAP25, synaptosomal-associated protein 25 kDa; SNARE, soluble NSF attachment protein receptors.

Figure 2

Arachidonic acid triggers a rapid change in syntaxin 1 intrinsic fluorescence. (A) Fluorescence spectra of syntaxin 1, SNAP25 and synaptobrevin±arachidonic acid (AA) with excitation at 280 nm. The peaks reflect either tyrosine (syntaxin 1; Stx1) or tryptophan fluorescence (SNAP25 and synaptobrevin; Syb). (B) Decrease in syntaxin 1 fluorescence at 308 nm occurs within 30 s of addition of arachidonic acid. (C) Neither saturated fatty acids nor lysophosphatidylcholine (LysoPC) affect syntaxin 1 structure in the same way as arachidonic acid. SNAP25, synaptosomal-associated protein 25 kDa.

Figure 3

Interaction of native Munc18 with syntaxin 1 persists after arachidonic-acid-induced SNAP25 engagement. (A) GST–syntaxin 1 (GST–Stx1), on glutathione beads, binds readily to bovine brain Munc18 and this association is not disrupted by 100 μM arachidonic acid (AA) or 1 M NaCl; 30 min reactions. Coomassie-stained gel. (B) AA (100 μM) allows SNARE assembly with an equimolar syntaxin 1–Munc18 complex. Coomassie-stained gel. (C) Incubation of GST–SNAP25 on glutathione beads with Stx1–Munc18 binary complex, in the presence of 100 μM AA, leads to pull-down of both syntaxin 1 and Munc18. Coomassie-stained gel. (D) Munc18 co-purifies with syntaxin 1–SNAP25 from brain extract (load) during preparative affinity chromatographies (AC) using anti-Munc18 or anti-SNAP25 BrCN beads. The control was BrCN Sepharose beads alone. Note the presence of SNAP25 in Munc18 AC and of Munc18 (red asterisk) in SNAP25 AC. Coomassie-stained gels. Representative peptides matching the Munc18 mass spectrometry profile are shown. (E) AA (200 μM) does not disrupt the native link between Munc18 and syntaxin 1–SNAP25 in synaptic membranes as judged by anti-Munc18 immunoprecipitation (IP) and western immunoblotting. (F) Syntaxin 1 presence on GST–SNAP25 beads (left) (Coomassie-stained gel) allows Munc18 pull-down from bovine brain cytosol. Munc18 was identified by western immunoblotting (right). GST, glutathione S-transferase; SNAP25, synaptosomal-associated protein 25 kDa; SNARE, soluble NSF attachment protein receptors; Syb, synaptobrevin.

Figure 4

Arachidonic acid is able to activate syntaxin 3 in the presence of Munc18. (A) GST–syntaxin 3, on glutathione beads, binds to Munc18-1 from bovine brain cytosol (top). Syntaxin 3–Munc18 association is not disrupted by 100 μM arachidonic acid but is sensitive to 1 M NaCl, indicating an electrostatic interaction (bottom). Coomassie-stained gels. GST, glutathione S-transferase. (B) Syntaxin 3, in equimolar complex with Munc18, is activated for SNARE assembly by 100 μM arachidonic acid (AA) and docosahexaenoic acid (DHA, C22:6). Coomassie-stained gel. (C) Schematic diagram showing the effect of AA on the syntaxin–Munc18 binary complex. AA allows SNAP25 engagement by syntaxin without Munc18 dissociation. SNAP25, synaptosomal-associated protein 25 kDa; Stx, syntaxin.