Interaction between Stoned and Synaptotagmin-1 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-DK) reduced MBP-C2B binding while the C-terminal tail deletion (GST-DW) 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-DK or GST-DW is normalized against MBP-C2B bound to GST-C2B. Each value represents mean 6 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 6 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 6 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. doi:10.1371/journal.pone.0038822.g004 Upon Ca2+ binding, SYT-1 was reported to undergo a confor- mational change that protects SYT-1 against trypsin and chymotrypsin digestion in vitro [37]. Ca2+ has also promoted homo-oligomerization of SYT-1 and/or hetero-oligomerization with other synaptotagmins [37]. Studies using mutations that affect Ca2+ dependent oligomerization, such as Y311N, showed a partial inhibition for internalization of SYT-1 in PC12 cells [24] while the corresponding mutation in Drosophila (AD3) alters the rate of exocytosis [21]. The AD3 SYT-1 is still capable of binding to SNARE complexes, synprint and AP-2, which implies that the mutation does not cause a complete loss of function [7]. Hence the docked vesicles in AD3 flies require higher Ca2+ concentration to undergo exocytosis [7], suggesting that defects in Ca2+ dependent oligomerization renders C2B SYT-1 inefficient for exocytosis. It was reported that Ca2+-triggered SYT-1 clustering is via the C2B domain and is required for exocytosis [37]. In a potential endocytic model that includes STNB, the oligomerized SYT-1 that has led to exocytosis, could then be a target for STNB binding, and STNB, in turn, could recruit dynamin via the intersectin DAP-160 [34]. This would create what amounts to PLoS ONE | www.plosone.org 7 June 2012 | Volume 7 | Issue 6 | e38822