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Munc18a does not alter fusion rates mediated by neuronal SNAREs, synaptotagmin, and complexin.

Zhang Y, Diao J, Colbert KN, Lai Y, Pfuetzner RA, Padolina MS, Vivona S, Ressl S, Cipriano DJ, Choi UB, Shah N, Weis WI, Brunger AT - J. Biol. Chem. (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: From the Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, Structural Biology, and Photon Science and.

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Related in: MedlinePlus

Effect of Munc18a on Ca2+-triggered fusion and spontaneous fusion between SV and PM vesicles after 30-min incubation of PM vesicles with Munc18a.A–F, histograms of Ca2+-triggered fusion events, without Munc18a or with Munc18a or Munc18a(S306E/S313E), using PM vesicles with reconstituted full-length Stx1a or Stx1a(10–288), as specified in the panels. All experiments were performed in the presence of 2 μm complexin. The histograms were calculated with a bin size of 1 s, normalized by the total of fusion events observed upon Ca2+ injection, and fitted to two-exponential decay functions (solid lines). The synchronization time constants (faster time constant of the fitted two-exponential fit) are shown in G. Spontaneous fusion events were observed for 1 min before Ca2+-injection and shown in H. Error bars in G, 1σ errors computed from the covariance matrix during least square fitting using a Levenberg-Marquardt technique; error bars in H, S.E. values over at least eight experiments.
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Figure 1: Effect of Munc18a on Ca2+-triggered fusion and spontaneous fusion between SV and PM vesicles after 30-min incubation of PM vesicles with Munc18a.A–F, histograms of Ca2+-triggered fusion events, without Munc18a or with Munc18a or Munc18a(S306E/S313E), using PM vesicles with reconstituted full-length Stx1a or Stx1a(10–288), as specified in the panels. All experiments were performed in the presence of 2 μm complexin. The histograms were calculated with a bin size of 1 s, normalized by the total of fusion events observed upon Ca2+ injection, and fitted to two-exponential decay functions (solid lines). The synchronization time constants (faster time constant of the fitted two-exponential fit) are shown in G. Spontaneous fusion events were observed for 1 min before Ca2+-injection and shown in H. Error bars in G, 1σ errors computed from the covariance matrix during least square fitting using a Levenberg-Marquardt technique; error bars in H, S.E. values over at least eight experiments.

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).


Munc18a does not alter fusion rates mediated by neuronal SNAREs, synaptotagmin, and complexin.

Zhang Y, Diao J, Colbert KN, Lai Y, Pfuetzner RA, Padolina MS, Vivona S, Ressl S, Cipriano DJ, Choi UB, Shah N, Weis WI, Brunger AT - J. Biol. Chem. (2015)

Effect of Munc18a on Ca2+-triggered fusion and spontaneous fusion between SV and PM vesicles after 30-min incubation of PM vesicles with Munc18a.A–F, histograms of Ca2+-triggered fusion events, without Munc18a or with Munc18a or Munc18a(S306E/S313E), using PM vesicles with reconstituted full-length Stx1a or Stx1a(10–288), as specified in the panels. All experiments were performed in the presence of 2 μm complexin. The histograms were calculated with a bin size of 1 s, normalized by the total of fusion events observed upon Ca2+ injection, and fitted to two-exponential decay functions (solid lines). The synchronization time constants (faster time constant of the fitted two-exponential fit) are shown in G. Spontaneous fusion events were observed for 1 min before Ca2+-injection and shown in H. Error bars in G, 1σ errors computed from the covariance matrix during least square fitting using a Levenberg-Marquardt technique; error bars in H, S.E. values over at least eight experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400359&req=5

Figure 1: Effect of Munc18a on Ca2+-triggered fusion and spontaneous fusion between SV and PM vesicles after 30-min incubation of PM vesicles with Munc18a.A–F, histograms of Ca2+-triggered fusion events, without Munc18a or with Munc18a or Munc18a(S306E/S313E), using PM vesicles with reconstituted full-length Stx1a or Stx1a(10–288), as specified in the panels. All experiments were performed in the presence of 2 μm complexin. The histograms were calculated with a bin size of 1 s, normalized by the total of fusion events observed upon Ca2+ injection, and fitted to two-exponential decay functions (solid lines). The synchronization time constants (faster time constant of the fitted two-exponential fit) are shown in G. Spontaneous fusion events were observed for 1 min before Ca2+-injection and shown in H. Error bars in G, 1σ errors computed from the covariance matrix during least square fitting using a Levenberg-Marquardt technique; error bars in H, S.E. values over at least eight experiments.
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).

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.

View Article: PubMed Central - PubMed

Affiliation: From the Departments of Molecular and Cellular Physiology, Neurology and Neurological Sciences, Structural Biology, and Photon Science and.

Show MeSH
Related in: MedlinePlus