Interaction between Stoned and Synaptotagmin-1 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 6 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 6 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. doi:10.1371/journal.pone.0038822.g001 are subsequently washed in EGTA-containing buffer (Fig. 1B, n = 3, p,0.05). STNB reverts to the level found when binding is carried out in the presence of EGTA. However, STNA binding is reduced but not abolished, suggesting that when bound, the dissociation of STNA from the C2B domain is less sensitive to the absence of Ca2+ than STNB. Ca2+ was also shown to effect the STNA/STNB interaction with the C2B domain at physiologically relevant Ca2+ levels. The interaction for STNA and STNB with C2B SYT-1 is achieved at Ca2+ concentration as low as 100– 200 mM free Ca2+ (data not shown). Phospholipids abolish the binding of STNB to C2B SYT-1 A recent study reported that m2 cannot replace the function of the mHD of STNB in vivo [34], and that the binding conditions of m2 of AP-2 to the C2B domain require Ca2+ and phosphatidyl serine (PS) [20]. We have investigated whether the binding of the mHD of STNB and the CCD of STNA to C2B SYT-1 is also PLoS ONE | www.plosone.org 3 June 2012 | Volume 7 | Issue 6 | e38822