Interaction between Stoned and Synaptotagmin-1 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-DK) 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-DK fusion proteins in Ca2+ buffer. The amount of bound MBP-Stoned proteins to GST-DK is normalized against the binding to GST-C2B. Each value represents mean 6 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-DH and GST-DW is normalized against MBP-Stoned proteins bound to GST-C2B. Experiments were done 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 stained of GST proteins of the respective blots as loading control. doi:10.1371/journal.pone.0038822.g003 STNB binding (Figure 3D). In the case of STNA, the binding to GST-DW increased over threefold (n = 3, p,0.05), and for STNB over fivefold (n = 4, p,0.05) as compared to the equivalent wild- type construct (Figure 3D). Deletion of both helices and the terminal b-strand (GST-DH) also increased the binding of MBP- STNB and MBP-STNA to a similar extent in comparison with the GST-DW fusion protein. These data indicate that the helices and the final b-strand, containing the WHTL motif, present in the C- terminal region of the C2B domain do not comprise the binding targets for the Stoned proteins, but must represent an ‘‘inhibitory component’’ of the C2B domain that affects the binding of both STNA and STNB. Perhaps there may be additional factors that interact with the C-terminal region of the C2B domain to regulate the binding of the Stoned proteins to SYT-I. In vitro studies using a GST-C2A-C2B domain, in which the polyK residues are deleted or mutated to alanines, showed an abolition of C2B oligomerization [19,37]. The C2B oligomeriza- tion is thought to regulate vesicle internalization, as alanine substitution of the oligomerization site (polyK to polyA) reduces the extent of internalization of SYT-1 in PC12 cells [24]. The C2A-C2B SYT-1 is also shown unable to oligomerize in the presence of Triton-X 100 treated phospholipids mixture contain- ing PS [25]. As the Stoned binding is reduced by PS containing phospholipids miscelles (Figure 2), these data suggest that the reduction in binding of Stoned proteins might be due to disruption of oligomerization of C2B SYT-1. We tested whether any of the truncated Drosophila C2B constructs affected their Ca2+-dependent oligomerization. Un- surprisingly, the wild-type C2B domain was found to self-associate, PLoS ONE | www.plosone.org 5 June 2012 | Volume 7 | Issue 6 | e38822