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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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Bio-layer interferometry experiments of Munc18a and Munc18a(S306E/S313E) binding to syntaxin-1A(1–265) or syntaxin-1A(25–265) constructs that had been surface-immobilized with a C-terminal biotin tag. Sensograms are fit to a 1:1 binding model (black lines). Specified concentrations of Munc18a were used. The dissociation constant of wild type Munc18a to Stx1a(1–265) is 4.5 nm (A), that of Munc18a(S306E/S313E) to Stx1a(1–265) is 75 nm (B), that of Munc18a to Stx1a(25–265) is 44 nm (C), and that of Munc18a(S306E/S313E) to Stx1a(25–265) is 1,400 nm (D). Note that binding of Munc18a to N-terminally truncated syntaxin-1A should not be affected by the different constructs (Stx1a(25–265) versus Stx1a(10–288)) used in bio-layer interferometry binding experiments and in the fusion and vesicle association experiments (see the supplemental material in Ref. 31).
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Figure 9: Bio-layer interferometry experiments of Munc18a and Munc18a(S306E/S313E) binding to syntaxin-1A(1–265) or syntaxin-1A(25–265) constructs that had been surface-immobilized with a C-terminal biotin tag. Sensograms are fit to a 1:1 binding model (black lines). Specified concentrations of Munc18a were used. The dissociation constant of wild type Munc18a to Stx1a(1–265) is 4.5 nm (A), that of Munc18a(S306E/S313E) to Stx1a(1–265) is 75 nm (B), that of Munc18a to Stx1a(25–265) is 44 nm (C), and that of Munc18a(S306E/S313E) to Stx1a(25–265) is 1,400 nm (D). Note that binding of Munc18a to N-terminally truncated syntaxin-1A should not be affected by the different constructs (Stx1a(25–265) versus Stx1a(10–288)) used in bio-layer interferometry binding experiments and in the fusion and vesicle association experiments (see the supplemental material in Ref. 31).

Mentions: Phosphorylation of Munc18a greatly reduces interaction with syntaxin-1A (41), and a phosphorylation-mimicking mutant Unc-18(S322E) of Caenorhabditis elegans Unc-18 (S322E) reduces binding to syntaxin-1A. However, the role of phosphorylation in neurons was found to be relatively subtle; the two phosphorylation sites in Munc18a appear to be important for post-tetanic potentiation (59). In our system, similar to wild type Munc18a, the phosphorylation mimic mutant of Munc18a(S306E/S313E) had no effect on the normalized Ca2+-triggered fusion histograms (Fig. 1, A–F), the synchronization time constant (Fig. 1G), or the probability of spontaneous fusion (Fig. 1H), regardless of whether full-length Stx1a or the N-terminally truncated Stx1a(10–288) fragment were used. The phosphorylation mimic Munc18a(S306E/S313E) also reduced vesicle association but to a lesser degree (Fig. 7A). The lessened effect on SV-PM vesicle association by the phosphorylation mimic is correlated with its reduced affinity to syntaxin-1A compared with wild type Munc18a, as determined by bio-layer interferometry experiments (Fig. 9). Thus, these experiments further corroborate that the sequestration of syntaxin-1A by Munc18a is related to the interaction between Munc18a and syntaxin-1A.


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)

Bio-layer interferometry experiments of Munc18a and Munc18a(S306E/S313E) binding to syntaxin-1A(1–265) or syntaxin-1A(25–265) constructs that had been surface-immobilized with a C-terminal biotin tag. Sensograms are fit to a 1:1 binding model (black lines). Specified concentrations of Munc18a were used. The dissociation constant of wild type Munc18a to Stx1a(1–265) is 4.5 nm (A), that of Munc18a(S306E/S313E) to Stx1a(1–265) is 75 nm (B), that of Munc18a to Stx1a(25–265) is 44 nm (C), and that of Munc18a(S306E/S313E) to Stx1a(25–265) is 1,400 nm (D). Note that binding of Munc18a to N-terminally truncated syntaxin-1A should not be affected by the different constructs (Stx1a(25–265) versus Stx1a(10–288)) used in bio-layer interferometry binding experiments and in the fusion and vesicle association experiments (see the supplemental material in Ref. 31).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: Bio-layer interferometry experiments of Munc18a and Munc18a(S306E/S313E) binding to syntaxin-1A(1–265) or syntaxin-1A(25–265) constructs that had been surface-immobilized with a C-terminal biotin tag. Sensograms are fit to a 1:1 binding model (black lines). Specified concentrations of Munc18a were used. The dissociation constant of wild type Munc18a to Stx1a(1–265) is 4.5 nm (A), that of Munc18a(S306E/S313E) to Stx1a(1–265) is 75 nm (B), that of Munc18a to Stx1a(25–265) is 44 nm (C), and that of Munc18a(S306E/S313E) to Stx1a(25–265) is 1,400 nm (D). Note that binding of Munc18a to N-terminally truncated syntaxin-1A should not be affected by the different constructs (Stx1a(25–265) versus Stx1a(10–288)) used in bio-layer interferometry binding experiments and in the fusion and vesicle association experiments (see the supplemental material in Ref. 31).
Mentions: Phosphorylation of Munc18a greatly reduces interaction with syntaxin-1A (41), and a phosphorylation-mimicking mutant Unc-18(S322E) of Caenorhabditis elegans Unc-18 (S322E) reduces binding to syntaxin-1A. However, the role of phosphorylation in neurons was found to be relatively subtle; the two phosphorylation sites in Munc18a appear to be important for post-tetanic potentiation (59). In our system, similar to wild type Munc18a, the phosphorylation mimic mutant of Munc18a(S306E/S313E) had no effect on the normalized Ca2+-triggered fusion histograms (Fig. 1, A–F), the synchronization time constant (Fig. 1G), or the probability of spontaneous fusion (Fig. 1H), regardless of whether full-length Stx1a or the N-terminally truncated Stx1a(10–288) fragment were used. The phosphorylation mimic Munc18a(S306E/S313E) also reduced vesicle association but to a lesser degree (Fig. 7A). The lessened effect on SV-PM vesicle association by the phosphorylation mimic is correlated with its reduced affinity to syntaxin-1A compared with wild type Munc18a, as determined by bio-layer interferometry experiments (Fig. 9). Thus, these experiments further corroborate that the sequestration of syntaxin-1A by Munc18a is related to the interaction between Munc18a and syntaxin-1A.

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