Munc18a does not alter fusion rates mediated by neuronal SNAREs, synaptotagmin, and complexin.
Bottom Line: Moreover, a phosphorylation mimic mutant of Munc18a with reduced affinity to syntaxin-1A results in less reduction of vesicle association.In summary, Munc18a does not directly affect fusion, although it has an effect on the t-SNARE complex, depending on the presence of other factors and experimental conditions.Our results suggest that Munc18a primarily acts at the prefusion stage.
Affiliation: From the Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, Structural Biology, and Photon Science and.Show MeSH
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Mentions: The N-terminally truncated construct of syntaxin-1A (Stx1a (10–288)) was expressed with an N-terminal thrombin-cleavable glutathione S-tranferase tag from plasmid pGEX-KG (49). After induction with 0.1 mm IPTG at A600 of 0.8, the proteins were expressed overnight at 16 °C at 100 rpm in Luria broth in BL21 (DE3) E. coli cells (Novagen, EMD Chemicals, Gibbstown, NJ). Cell pellets from 1 liter of culture were suspended in 40 ml of sodium phosphate, pH 8, 1 mm dithiothreitol, 0.2% Triton X-100, 1% dodecylmaltoside, and 0.2 mm PMSF supplemented with Complete Protease Inhibitor Mixture tablets (Roche Applied Science), and the cells were disrupted by sonication at 5/15-s on/off pulse for 5 min at 50% power using a Sonicator Ultrasonic Processor XL-2020 (Misonix, Farmingdale, NY). Inclusion bodies were removed by a 10-min 15,000-rpm spin in a JA-20 (Beckman Coulter) rotor and further cleared by centrifugation at 40,000 rpm for 35 min in a Ti-45 (Beckman Coulter) rotor. The supernatant was incubated for 2 h with a 1.5-ml slurry of glutathione-Sepharose 4 Fast Flow beads (GE Healthcare) that was pre-equilibrated in the same buffer. The beads were then collected in a column and washed with 80 column volumes of buffer containing 50 mm sodium phosphate, pH 8, 1 mm dithiothreitol, and 0.2% Triton X-100 and then with 30 column volumes of buffer containing 40 mm Tris-HCl, pH 8, 150 mm NaCl, 1 mm dithiothreitol, and 0.8% OG. The beads were then incubated for 90 min at ambient temperature with 100 units of bovine α-thrombin (Hematologic Technologies, Essex Junction, VT) in buffer containing 40 mm Tris-HCl, pH 8, 150 mm NaCl, 1 mm dithiothreitol, and 3% OG. The supernatant containing cleaved ΔN syntaxin(10–288) was flowed off the column, after which the thrombin was quenched with 1 mm PMSF. Although the GST-syntaxin-1A construct was designed to include the N terminus of syntaxin-1A, the application of thrombin proteolysis also removed nine additional residues at the N terminus as determined by Edman sequencing. Removal of these nine N-terminal residues has no significant effect on fusion efficiency and kinetics for spontaneous and Ca2+-triggered fusion with neuronal SNAREs, synaptotagmin-1, and complexin-1 (Fig. 1, G and H).
Affiliation: From the Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, Structural Biology, and Photon Science and.