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Figure 1. Syx and CSYS are targets for CK2 phosphorylation. (A) Domain structure of Syx and the CSYS FRET probe. (B) CSYS is a target for CK2 phosphorylation similarly to native Syx. (a) Native PC12 cells were immunoprecipitated with anti-Syx antibody and immunoblotted either with anti-Syx antibody (IB-Syx; upper panel) or with Syx S14 phospho-specific antibody (IB- S14 phos; lower panel). Brain membranes (BM) were loaded as a control. (b) Following immunoprecipitation with anti-YFP antibody, proteins of PC12 cells transfected with CSYS were subjected to Western blot analysis with either anti-Syx antibody (IB-Syx; upper panel) or with Syx S14 phospho-specific antibody (IB-S14 phos; lower panel). Molecular markers are shown on the left. (c) Xenopus oocytes expressing CSYS and Syx proteins were immunoprecipitated with anti-Syx antibody (35S Syx; left panel) and immunoblotted with Syx S14 phospho-specific antibody (IB-S14 phos; right panel).
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Figure 2. The phospho-null mutant undergoes CDO that does not require intact 5RK. (A) Domain structure of the CSYS mutants; CSYS;S14A and CSYS-5RK/A;S14A. Red font highlights important residues (B) The phospho-null mutant CSYS;S14A undergoes CDO. (a) The reduction in the FRET ratio of the phospho-null mutant CSYS;S14A in response to hK depolarization is similar to that of CSYS (black squares, n = 30 cells, and grey diamonds n = 32 cells, respectively; 3 experiments). (b) Part of the FRET ratio reduction of CSYS;S14A is blocked by Cd and represents CDO. Addition of 200 µM Cd to both control and hK solutions resulted in a smaller reduction in the FRET ratio (white squares, n = 22 cells, and black squares, n = 26 cells; 2 experiments; F(79,6083) = 15.166, p < 0.0001, ANOVA with repeated measures). (C) CDO in the phospho-null mutant does not require intact 5RK. Despite the 5RK/A mutation, the reduction in FRET ratio of CSYS-5RK/A;S14A in response to hK depolarization was similar to that of CSYS;S14A (grey circles, n = 39, and black squares, n = 44, respectively; 5 experiments) and was smaller in the presence of Cd (white circles n = 40, 5 experiments; F(79,3397) = 5.411, p < 0.0001, ANOVA with repeated measures). Gray background is 200 sec stimulation with high K+.
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Figure 3. The phospho-mimetic mutant undergoes CDO that requires intact 5RK. (A) Domain structure of the phospho-mimetic CSYS mutants, CSYS;S14E and CSYS-5RK/A;S14E. Red font highlights important residues. (B) The reduction in the FRET ratio in response to hK depolarization of CSYS;S14E was similar to that of CSYS (a); black triangles, n = 18, and grey diamonds n = 20, respectively; 2 experiments) and included CDO that requires intact 5RK, similarly to CSYS, as it was smaller upon insertion of the 5RK/A mutation (b); black triangles n = 18, and grey triangles, n = 20; 2 experiments; F(79,2844) = 4.331, p < 0.0001, ANOVA with repeated measures). Gray background is 200 sec stimulation with high K+.
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Figure 4. Neutralization of the N-terminal CK2 recognition site in CSYS mimics the effect captured by the phospho-null mutant, eliminating the requirement of 5RK for CDO. (A) Domain structures of the CSYS mutants, CSYS;5D/A and CSYS-5RK/A;5D/A. Red font highlights important residues. (B) The FRET ratio reduction in response to hK depolarization of CSYS;5D/A was similar to that of CSYS (grey triangles, n = 38 and black diamonds, n = 65, respectively; 7 experiments). (C) The FRET ratio reduction of CSYS-5RK/A;5D/A was larger than that of CSYS-5RK/A (a); grey diamonds, n = 35, and grey squares n = 34, respectively; 7 experiments; F(79,5214) = 5.576, p < 0.0001, ANOVA with repeated measures) and was smaller in the presence of Cd, representing CDO that does not require intact 5RK (b); white diamond, n = 39, and grey diamonds, n = 42, respectively; 3 experiments; F(79,5688) = 8.959, p < 0.0001, ANOVA with repeated measures).
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Figure 5. Inhibition of in vivo S14 phosphorylation eliminates the requirement of 5RK for CDO. (A) S14 phosphorylation is inhibited by TBB. (a) S14 phosphorylation of endogenous Syx in PC12 cells is reduced by TBB. Native Syx was immunoprecipitated with anti-Syx antibody and immunoblotted with either anti-Syx antibody (IB-Syx; upper panel) or with Syx S14 phospho-specific antibody (IB-S14 phos; middle panel). The extent of phosphorylation (ratio between the corresponding upper and middle intensities) was reduced by TBB (lower panel). Native brain membranes (BM) were loaded as a control. (b) S14 phosphorylation of CSYS-5RK/A expressed in PC12 cells is reduced by TBB. CSYS-5RK/A was immunoprecipitated from transfected PC12 cells with anti-YFP antibody and immunoblotted with either anti-Syx antibody (IB-Syx; upper panel) or with Syx S14 phospho-specific antibody (IB-S14 phos; middle panel). In the presence of TBB the extent of S14 phosphorylation was reduced (lower panel). Molecular weight markers are shown on the left. Native cells were used for control (c). (B) De-phosphorylation by TBB rescues CDO in CSYS-5RK/A. (a) Addition of TBB increased the FRET ratio reduction of cells expressing CSYS-5RK/A in response to hK depolarization (grey circles, n = 25, and black squares, n = 19; 2 experiments; F(79,3239) = 3.454, p < 0.0001, ANOVA with repeated measures) (b) In the presence of TBB the FRET ratio reduction was smaller upon addition of Cd (white circles n = 34, and grey circles n = 34, respectively; 3 experiments; F(79,5214) = 3.410, p < 0.0001, ANOVA with repeated measures).
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Figure 6. (A–C) A requirement for PIP2 is an intrinsic feature of CDO and is independent of S14 phosphorylation. Upon PIP2 hydrolysis by co-expressed PLCη2, the FRET ratio reduction in response to hK depolarization was reduced in cells expressing each of the phospho-null mutants, CSYS;S14A (A); grey squares, n = 50, and black squares, n = 42, respectively; five experiments; F(79,6794) = 8.167, p < 0.0001, ANOVA with repeated measures) and CSYS-5RK/A;S14A (B); black diamonds, n = 16 and grey diamonds, n = 20, respectively; two experiments; F(79,2686) = 5.343, p < 0.0001, ANOVA with repeated measures). The FRET ratio reduction of CSYS-5RK/A;S14A in the presence of PLCη2 was insensitive to the addition of Cd, indicating that PLCη2 inhibited CDO (C); white diamonds, n = 21 and black diamonds, n = 23, respectively; two experiments). (D) The conformational transition of the CSYS phospho-null mutant does not involve Syb2. Syb2 cleavage by TeTx-LC did not have any effect on the FRET ratio reduction in response to hK depolarization of CSYS;S14A (black squares, n = 23 and grey squares, n = 20, respectively; three experiments).
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Figure 7. The phospho-null mutation abolishes Ca2+-triggered release and the enhanced spontaneous release conferred by the 5RK/A mutation. (A) CSYS;S14A (R), bearing the K253I mutation, is located in the PM region of PC12 cells and is resistant to cleavage by BoNT-C1. Upper panel: confocal images of PC12 cells demonstrating that CSYS;S14A (R) (iii) retained its PM distribution upon BoNT-C1 co-expression (iv), indicating its resistance to BoNT-C1, while CSYS;S14A (i) was located in the cytosol upon BoNT-C1 co-expression (ii). Scale bar: 5 mm. Lower panel: normalized fluorescence intensity profiles of the above cells indicating PM or cytosolic expression. The fluorescence profiles were determined from line scans (red lines, upper panel) taken from the outside to the middle of each cell. (B) Representative amperometric recordings of catecholamine release from cells transfected CSYS (R) or with CSYS;S14A (R), before and after hK depolarization. (C) Average number of spikes (a) and total charge release (b) for spontaneous (before hK) and evoked (after hK) release from PC12 cells co-transfected with BoNT-C and either CSYS (R) or CSYS;S14A (R) (t(14) = 2.16, * p = 0.048 (a) and t(14) = 2.84, * p = 0.013 (b), paired t-test). (D) Average number of spikes for spontaneous release from PC12 cells co-transfected with BoNT-C and either CSYS-5RK/A (R) or CSYS-5RK/A;S14A (R) (n = 11 and 8, respectively, * p = 0.044, Mann-Whitney Rank Sum test).
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Figure 8. Model of the interrelated roles of 5RK and S14 phosphorylation in Syx functioning during exocytosis. (A) Left panel, Syx (blue cylinders) maintains the closed conformation at release competent sites (green; release site) due to S14 phosphorylation by CK2. Prior to Ca2+ entry, 5RK (pink triangle) interacts with certain lipids and/or fusogenic proteins that enable 5RK to act as a fusion clamp that inhibits spontaneous release. Under these conditions, PIP2 is not likely to interact with Syx (possibly with the N terminus), because of hindrance by the negative charge of the phosphate group of phosphorylated S14. Middle and right panels, Sequential reactions in response to depolarization-induced intracellular Ca2+ elevation start with reorganization of the release site and an intramolecular rearrangement of Syx that draws the positive charges of 5RK and the negative charges of phosphorylated S14 into close proximity, resulting in electrostatic-charge shielding (grey shade) of the phosphate group (middle). The consequent formation of the PIP2–Syx interaction (orange) enables Syx to undergo CDO and to promote Ca2+-triggered release (right). (B) Left panel, the S14-phosphorylated 5RK/A mutant lacks the 5RK clamp and exhibits enhanced spontaneous release. Middle and right panels, In response to depolarization-induced intracellular Ca2+ elevation there is no charge shielding of the phosphate group so that PIP2 cannot interact with Syx, consequently Syx cannot undergo CDO and promote Ca2+-triggered release. (PM; plasma membrane).
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