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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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Cytoplasmic SNARE complex formation starting from cytoplasmic, soluble t-SNAREs with and without Munc18a. The amount of cytoplasmic SNARE complex formed (A) and initial rate of SNARE complex formation (B) were determined using fluorescence anisotropy with synaptobrevin-2(1–96)-A488. SNARE complex formation from three starting states was compared: t-SNAREs in the absence of Munc18a (red); t-SNAREs with sequential addition of Munc18a (blue); and t-SNAREs in a preformed complex with Munc18a, where Munc18a, Stx1a(1–267), and SNAP-25 were used in equal proportions (green). We note that protein constructs and molar ratios in this work differ from those in Ref. 70, so the rates of SNARE complex formation cannot be compared directly.
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Figure 5: Cytoplasmic SNARE complex formation starting from cytoplasmic, soluble t-SNAREs with and without Munc18a. The amount of cytoplasmic SNARE complex formed (A) and initial rate of SNARE complex formation (B) were determined using fluorescence anisotropy with synaptobrevin-2(1–96)-A488. SNARE complex formation from three starting states was compared: t-SNAREs in the absence of Munc18a (red); t-SNAREs with sequential addition of Munc18a (blue); and t-SNAREs in a preformed complex with Munc18a, where Munc18a, Stx1a(1–267), and SNAP-25 were used in equal proportions (green). We note that protein constructs and molar ratios in this work differ from those in Ref. 70, so the rates of SNARE complex formation cannot be compared directly.

Mentions: Having established that Munc18a binds to the cytoplasmic (non-membrane anchored) t-SNARE complex, we monitored cytoplasmic ternary SNARE complex formation in the absence or presence of Munc18a. The kinetics of ternary SNARE complex formation was measured by fluorescence anisotropy, by adding fluorescently labeled synaptobrevin-2 cytoplasmic domain (synaptobrevin-2(1–96)-Alexa488) to a preformed cytoplasmic t-SNARE complex consisting of Stx1a(1–267) and SNAP-25. An increase in anisotropy, which is positively correlated with an increase of molecular mass, indicates the formation of SNARE complex. Using a 1:1 molar ratio of Stx1a(1–267) and SNAP-25 present at 6–100-fold molar excess over synaptobrevin-2(1–96)-Alexa488, we monitored the rate of ternary SNARE complex formation under three conditions (Figs. 5 and 6): 1) no Munc18a (Fig. 5, red line); 2) incubating Munc18a with the t-SNARE complex for 0.5–2 h prior to initiating the complex formation reaction (Fig. 5, blue line); and 3) starting from a gel filtration-purified 1:1:1 complex of Munc18a·Stx1a(1–267)·SNAP-25 (Fig. 5, green line). As a control, we confirmed that adding Munc18a to Stx1a(1–267) before adding SNAP-25, using a 1:1:1 molar ratio of Munc18a·Stx1a(1–267)·SNAP-25 over a range of concentrations, inhibits neuronal SNARE complex formation (31).


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)

Cytoplasmic SNARE complex formation starting from cytoplasmic, soluble t-SNAREs with and without Munc18a. The amount of cytoplasmic SNARE complex formed (A) and initial rate of SNARE complex formation (B) were determined using fluorescence anisotropy with synaptobrevin-2(1–96)-A488. SNARE complex formation from three starting states was compared: t-SNAREs in the absence of Munc18a (red); t-SNAREs with sequential addition of Munc18a (blue); and t-SNAREs in a preformed complex with Munc18a, where Munc18a, Stx1a(1–267), and SNAP-25 were used in equal proportions (green). We note that protein constructs and molar ratios in this work differ from those in Ref. 70, so the rates of SNARE complex formation cannot be compared directly.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Cytoplasmic SNARE complex formation starting from cytoplasmic, soluble t-SNAREs with and without Munc18a. The amount of cytoplasmic SNARE complex formed (A) and initial rate of SNARE complex formation (B) were determined using fluorescence anisotropy with synaptobrevin-2(1–96)-A488. SNARE complex formation from three starting states was compared: t-SNAREs in the absence of Munc18a (red); t-SNAREs with sequential addition of Munc18a (blue); and t-SNAREs in a preformed complex with Munc18a, where Munc18a, Stx1a(1–267), and SNAP-25 were used in equal proportions (green). We note that protein constructs and molar ratios in this work differ from those in Ref. 70, so the rates of SNARE complex formation cannot be compared directly.
Mentions: Having established that Munc18a binds to the cytoplasmic (non-membrane anchored) t-SNARE complex, we monitored cytoplasmic ternary SNARE complex formation in the absence or presence of Munc18a. The kinetics of ternary SNARE complex formation was measured by fluorescence anisotropy, by adding fluorescently labeled synaptobrevin-2 cytoplasmic domain (synaptobrevin-2(1–96)-Alexa488) to a preformed cytoplasmic t-SNARE complex consisting of Stx1a(1–267) and SNAP-25. An increase in anisotropy, which is positively correlated with an increase of molecular mass, indicates the formation of SNARE complex. Using a 1:1 molar ratio of Stx1a(1–267) and SNAP-25 present at 6–100-fold molar excess over synaptobrevin-2(1–96)-Alexa488, we monitored the rate of ternary SNARE complex formation under three conditions (Figs. 5 and 6): 1) no Munc18a (Fig. 5, red line); 2) incubating Munc18a with the t-SNARE complex for 0.5–2 h prior to initiating the complex formation reaction (Fig. 5, blue line); and 3) starting from a gel filtration-purified 1:1:1 complex of Munc18a·Stx1a(1–267)·SNAP-25 (Fig. 5, green line). As a control, we confirmed that adding Munc18a to Stx1a(1–267) before adding SNAP-25, using a 1:1:1 molar ratio of Munc18a·Stx1a(1–267)·SNAP-25 over a range of concentrations, inhibits neuronal SNARE complex formation (31).

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