Ca2+ regulates the Drosophila Stoned-A and Stoned-B proteins interaction with the C2B domain of Synaptotagmin-1

Abstract

The dicistronic Drosophila stoned gene is involved in exocytosis and/or endocytosis of synaptic vesicles. Mutations in either stonedA or stonedB cause a severe disruption of neurotransmission in fruit flies. Previous studies have shown that the coiled-coil domain of the Stoned-A and the µ-homology domain of the Stoned-B protein can interact with the C2B domain of Synaptotagmin-1. However, very little is known about the mechanism of interaction between the Stoned proteins and the C2B domain of Synaptotagmin-1. Here we report that these interactions are increased in the presence of Ca(2+). The Ca(2+)-dependent interaction between the µ-homology domain of Stoned-B and C2B domain of Synaptotagmin-1 is affected by phospholipids. The C-terminal region of the C2B domain, including the tryptophan-containing motif, and the Ca(2+) binding loop region that modulate the Ca(2+)-dependent oligomerization, regulates the binding of the Stoned-A and Stoned-B proteins to the C2B domain. Stoned-B, but not Stoned-A, interacts with the Ca(2+)-binding loop region of C2B domain. The results indicate that Ca(2+)-induced self-association of the C2B domain regulates the binding of both Stoned-A and Stoned-B proteins to Synaptotagmin-1. The Stoned proteins may regulate sustainable neurotransmission in vivo by binding to Ca(2+)-bound Synaptotagmin-1 associated synaptic vesicles.

Figure 1. Ca²⁺ enhances the binding of the STNA and STNB proteins to the C2B domain of SYT-1.

Preincubation of nucleic acid free GST-C2B with 1mM CaCl₂ in Hepes binding buffer increased the MBP-STNB (n=6) and MBP-STNA (n=6) binding, compared to preincubation in 1 mM EGTA containing buffer. In control, neither MBP-STNA nor MBP-STNB binds to GST protein in the presence of 1 mM CaCl₂. The quantified MBP-Stoned proteins are normalized against the amount of eluted GST-C2B. The bound MBP-Stoned proteins in Ca²⁺ are normalized against MBP-Stoned proteins bound in EGTA buffer. Each value represents mean ± S.E.M. B. The STNA and STNB proteins, bound to GST-C2B in the presence of Ca²⁺, were subsequently washed with 1 mM EGTA-containing buffer. Washing in EGTA reduced the amount of bound MBP-STNB (n=3) and MBP-STNA (n=3) proteins, compared with equivalent washes in buffer containing 1 mM CaCl₂ (Ca²⁺ washing buffer). The bar graph represents the amount of quantified MBP-Stoned proteins. The quantified MBP-Stoned proteins are normalized against the amount of eluted GST-C2B. The bound MBP-Stoned proteins in EGTA buffer is normalized against MBP-Stoned proteins bound to GST-C2B in Ca²⁺buffer. Each value represents mean ± S.E.M. The upper panel of each Western Blot images is the MBP-Stoned proteins bound to GST-C2B and detected by anti-MBP antibody while the lower panel is the Ponceau Red staining of GST-C2B of the respective blots as loading control.

Figure 2. Addition of phospholipids mixture reduces the STNB binding to GST-C2B.

Binding of MBP STNA binding to GST-C2B is not affected by phospholipids (n=3). B. Phospholipids mixture containing phosphatidylserine (20% PE: phosphatidylethanolamine; 60% PC: phosphatidylcholine, 20% PS: phosphatidylserine) reduces the binding of MBP-STNB (n=4) to GST-C2B in comparison with binding in the presence of Ca2⁺ (*n=4, p<0.01) or PE:PC+ Ca2⁺ (**n=4, p<0.05) to the GST-C2B. “PE:PC” indicates 20%PE:80%PC. “PE:PC:PS” indicates 20%PE:60%PC: 20%PS. The quantified MBP-Stoned proteins are normalized against the amount of eluted GST-C2B. The bound MBP-Stoned proteins are normalized against MBP-Stoned proteins bound to GST-C2B in EGTA buffer. Each value represents mean ± S.E.M. The upper panel of each Western Blot images is the MBP-Stoned proteins bound to GST-C2B and detected by anti-MBP antibody while the lower panel is the Ponceau Red staining of GST-C2B of the respective blots as loading control.

Figure 3. Truncations of the C2B domain of SYT-1 affected STNA and STNB proteins binding.

A. Several deletion constructs of C2B were created in respect of important residues known to alter the SYT-1 properties. B. Amino acid sequence of C-terminal truncated constructs of SYT-1-C2B domain. C. Deletion of the N-terminal polylysine region (GST-ΔK) reduced MBP-STNA and MBP-STNB proteins binding, in comparison with binding to GST-C2B. The graph represents the amount of quantified MBP-STNA (n=3) and MBP-STNB (n=3) proteins normalized against the amount of eluted GST-C2B or GST-ΔK fusion proteins in Ca²⁺ buffer. The amount of bound MBP-Stoned proteins to GST-ΔK is normalized against the binding to GST-C2B. Each value represents mean ± S.E.M. D. Deletions of C-terminal C2B increased the MBP-STNA and MBP-STNB binding. The graphs represent the amount of quantified MBP-STNA (n=3) and MBP-STNB (n=3) proteins normalized against the amount of eluted GST fusion proteins in Ca²⁺ buffer. The amount of bound MBP-Stoned proteins to GST-ΔH and GST-ΔW is normalized against MBP-Stoned proteins bound to GST-C2B. Experiments were done in Ca²⁺ buffer. Each value represents mean ± S.E.M. The upper panel of each Western Blot images is the MBP-Stoned proteins bound to GST-C2B and detected by anti-MBP antibody while the lower panel is the Ponceau Red stained of GST proteins of the respective blots as loading control.

Figure 4. Ca² dependent oligomerization regulates STNA and STNB proteins binding to the C2B domain of SYT-1.

A. Truncation of C2B affects oligomerization. The N-terminal polylysine deletion (GST-ΔK) reduced MBP-C2B binding while the C-terminal tail deletion (GST-ΔW) increased MBP-C2B binding fourfold (n=3) in comparison with binding to GST-C2B. Independent experiments were done in Ca²⁺ buffer. The amount of quantified MBP proteins is normalized against the amount of eluted GST proteins. The amount of bound MBP-C2B to GST-ΔK or GST-ΔW is normalized against MBP-C2B bound to GST-C2B. Each value represents mean ± S.E.M. B. The D3,4N mutation abolished the effect of Ca² to regulate C2B oligomerization in comparison to GST-C2B in Ca² buffer (n=3, p<0.05). The amount of quantified MBP proteins is normalized against the amount of eluted GST proteins. The amount of bound MBP-C2B to GST-C2B or GST-D3,4N is normalized against MBP-C2B bound to GST-C2B in EGTA buffer. Each value represents mean ± S.E.M. C. The D3,4N mutation also reduced STNA (*n=3, p<0.05) and STNB (**n=5, p<0.01) binding by comparison with binding to GST-C2B in Ca² buffer. In the presence of Ca²⁺ buffer, the D3,4N mutation significantly increased the MBP-STNB binding in comparison to GST-D3,4N in EGTA buffer (***n=5, p<0.05). The amount of quantified MBP proteins is normalized against the amount of eluted GST proteins. The amount of bound MBP-Stoned proteins to GST-C2B or GST-D3,4N is normalized against MBP-Stoned proteins bound to GST-C2B in EGTA buffer. Each value represents mean ± S.E.M. The upper panel of each Western Blot image shows the MBP tagged proteins bound to GST tagged proteins and detected by anti-MBP antibody; while the lower panel is the Ponceau Red staining of GST proteins of the respective blots as loading control.

Figure 5. The effect of pC2B1 peptide on STNA and STNB proteins binding and C2B self-association.

The experiments were done in the presence of 1mM CaCl₂. A. Addition of 10 µM pC2B1 reduced the MBP-STNA and abolished MBP-STNB binding to GST-C2B domain. B. Increasing concentration of pC2B1 in a dose dependent manner (0 to 4 µM) outcompeted MBP-STNB (0.25 µM) binding to 1 µM GST-C2B. The graph represents the amount of quantified MBP-STNA and MBP-STNB proteins normalized against the amount of eluted GST-C2B and the binding of MBP-Stoned in 0 µM pC2B1. Each value represents mean ± S.E.M (n=3). C. The pC2B1 peptide also reduced C2B SYT-1 oligomerization. The graph represents the amount of quantified MBP-C2B normalized against the amount of eluted GST- C2B. Each value represents mean ± S.E.M (n=3). The upper panel of each Western Blot image shows the MBP tagged proteins bound to GST-C2B and detected by anti-MBP antibody; while the lower panel is the Ponceau Red staining of GST proteins of the respective blots as loading control.