Interaction between Stoned and Synaptotagmin-1 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 mM 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 mM) outcompeted MBP-STNB (0.25 mM) binding to 1 mM 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 mM pC2B1. Each value represents mean 6 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 6 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. doi:10.1371/journal.pone.0038822.g005 a Ca2+-dependent endocytic complex. However, a previous study has shown that STNB can be found bound to synaptic vesicles, via SYT-1, prior to exocytosis [17], that is, prior to the Ca2+ influx that might trigger SYT-1 oligomerization and the coupling of excitation and vesicle fusion [37]. Either this bound STNB reflects the low level of oligomerization of SYT-1 in the absence of Ca2+, or that other factors, such as those that might alter the structure of the C-terminal region of SYT-I C2B, are playing a role in potentiating the binding of STNB to SYT-1 even in the absence of Ca2+. Certainly there is no STNB protein in the soluble fraction from fly head extracts [17], suggesting that all STNB is in a bound form, and although some is certainly is, perhaps not all is bound to SYT-1. The D3,4N mutation in transgenic flies results in a reduction in the rate of endocytosis of synaptic vesicles [21], this may be due to a failure of this mutant SYT-I to interact with Stoned proteins. Another mutation which gives constitutive dimerization, the D3,4N mutation [37], was incapable to restore internalization in CHO cells [24]. The D3,4N mutation did not only shows a reduction in the rate of endocytosis in Drosophila, but also exocytosis defect by decrease evoked transmitter release and reduce in apparent Ca2+ affinity for synaptic transmission [43,44]. Indeed, similar to these synaptotagmin mutant flies, the stoned mutants showed alterations in both spontaneous and evoked release at larval NMJ and severe neurotransmission defect that may lead to embryonic lethality [29,45], as well as depletion of synaptic vesicle and increase of membrane recycling intermediate that might be due to mislocalization of synaptotagmin during endocytosis [45]. In contrast with m2 and SYT-1 interaction [20], the binding of Stoned proteins do not require phospholipids. Studies using Folch liposome has shown that D3,4N mutant are not able to induce a close proximity membrane curvature, which may be a prerequisite for SNARE mediated membrane fusion [27]. A synthesized peptide consist of the 3rd loop of C2B SYT-1 can outcompete the m-homology domain of STNB (our data), suggesting the interaction of STNB to SYT-1 is mediated by a region in loop 3 of C2B domain. Thus, it is an attractive hypothesis that STNB may act as an inhibitor for membrane PLoS ONE | www.plosone.org 8 June 2012 | Volume 7 | Issue 6 | e38822