Two other regions of the C2B domain have been implicated in retrieval of SYT-1 from the plasma membrane. It has been shown that a tryptophan containing motif, present in the last b-strand of the C2B domain (WHTL), can act as an internalization signal in chimeric proteins expressed in PC12 cells [24], and the sub- stitution of asparagine residues for two aspartates in one of the Ca2+-binding loops of the C2B domain, when present in transgenic Drosophila, results in the reduction of SV endocytosis to the equivalent of SYT-1 mutant allele [21]. The WHXL motif in b-strand 8 of C2B is reported to mediate internalization to a similar extent to that seen in the C-terminal tail construct where helix A, b-strand 8 and helix B are deleted [26]. The C-terminus, in a chimeric construct triggered internalization; however, in- vestigation of the C2B structure shows that the W404 residue is only partially exposed [26]. In order to allow internalization, there should be a conformational change in the C2B domain that would allow recognition of the WHXL motif. Whether change in conformation of the C2B domain is caused by interaction with other proteins, such as Stoned proteins, or by other mechanisms, is still unclear [26]. Mutation of the C2B residues Asp416 and Asp418 to asparagines (in D3,4N flies) caused a defect in exocytosis due to the disruption of bound Ca2+. The observed evoked transmitter release is decreased by 95% and the apparent Ca2+ affinity for synaptic transmission is also reduced [21]. However, these defects in exocytosis may be caused by depletion of recycled vesicles, as this mutation also reduces the rate of SV endocytosis to the equivalent of that of a SYT-1 mutant allele. The slower rate of endocytosis in D3,4N flies, is different from that in SYT-1 flies, as D3,4N flies can be rescued by increasing extracellular Ca2+, suggesting that Ca2+ coordination is important for regulating the rate of endocytosis [21]. In contrast to the polyK mutation, no significant alteration in vesicle size was observed in D3,4N flies [21]. Much research has focused on the role of the AP-2 complex in SYT-1-induced SV endocytosis. Phospholipids have been shown to interact with SYT-1 [27,28], and it has been shown that the binding of m2 of AP-2 with the C2B domain of SYT-1 is mediated by the presence of a phospholipid mixture containing phospha- tidylserine (PS) [20]. Ca2+-triggered rearrangements between proteins and lipids are believed to cause the opening and dilation of a fusion pore which subsequently leads to the exocytic release of the vesicle contents. Ca2+ is postulated to first drive C2 domains membrane interactions, which result in a conformational change that triggers a subsequent C2B-mediated oligomerization step [25]. In Drosophila, mutations at the stoned locus are known to affect the rates of SV endocytosis and alter the presynaptic distribution of SYT-1, even though the distribution of endocytic proteins such as dynamin, a-adaptin and clathrin, are unaltered [29,30,31]. This Ca2+-mediated conformational change of SYT-1 may be impor- tant in mediating the interaction of the Stoned proteins with SYT- 1. The Drosophila stoned locus is dicistronic, encoding two polypep- tides, Stoned-A (STNA) and Stoned-B (STNB) [32,33]. Structur- ally STNA shows no homology to any protein outside of the Insecta, but contains an N-terminal lysine rich domain followed by a coiled coil region (CCD; coiled coil domain) and a C-terminal domain consisting of four repeats, each of which contains an aspartate-proline-phenylalanine (DPF) motif [33]. This DPF motif has been shown in other proteins to interact with the appendage domain of the a-adaptin subunit of AP-2. The STNB protein contains a C-terminal domain that shows homology to the m- subunit of AP-2 (mHD; m-Homology Domain) [32], and an extended N-terminal domain rich in proline and containing a series Interaction between Stoned and Synaptotagmin-1 of asparagine-proline-phenylalanine (NPF) motifs that are known targets for EH domain-containing proteins. We have shown that the N-terminal region of STNB has a high affinity for the Drosophila intersectin, DAP-160 [33,34]. The possible roles of the Drosophila STNA and STNB proteins have been primarily studied through their interactions with SYT-1 [17]. The binding of STNB to SV is via SYT-I, and appears to be prior to exocytosis, as the blockade of endocytosis in active synapses, using temperature-sensitive dynamin mutant shibire, does not prevent STNB-bound vesicles from being recovered, from, presumably, inactive synapses in these dark adapted flies [17]. In vitro studies have shown that STNB, and its mammalian homologue, stonin-2, binds via the mHD to the C2B domain of SYT-1, while STNA also binds to the C2B domain via its CCD [17,35]. Ca2+ may play a role in this interaction. When Drosophila heads are homogenized in the presence of Ca2+, STNB and SYT- 1 are found almost entirely in the plasma membrane fraction while in the absence of Ca2+, both STNB and SYT-1 are found in both plasma membrane and vesicle enriched fractions [17]. This suggests that Ca2+ may have an important role in the binding of the Stoned proteins to SYT-1. Exactly what the role might be for the Stoned proteins in SV recycling might be, is unclear, yet they would appear to be essential for normal SV recycling in the fly. We therefore continue with our study of these proteins and especially with their interaction with SYT-I. Here we report our investigations of the effect of Ca2+ and phospholipids on the binding of the CCD of STNA and the mHD of STNB to the purified recombinant C2B domain of SYT-1. We have found that Ca2+ greatly increases the binding of STNA and STNB to the C2B domain of SYT-1, that these interactions are primarily driven by a Ca2+-dependent self-association of the C2B domains of SYT-1 molecules and that the likely binding site for STNB resides within the Ca2+-binding loops of the dimeric or multimeric C2B protein domains. We have also observed a negative effect of phospholipids on the in vitro binding of STNB to the C2B domain of SYT-I. Results Interactions between STNA/STNB and the Synaptotagmin-1 C2B domain is Ca2+-dependent Figure 1A shows that the presence of Ca2+ considerably enhanced the binding of both MBP-STNB (MHD) and MBP- STNA (CCD) proteins to the GST SYT-1 C2B domain. For STNA this increase was 2.5 fold (n = 6, p,0.05) while for STNB it was 4 fold (n = 6, p,0.05). The level of binding of the MBP-STNA and MBP-STNB proteins to GST-C2A was slightly higher than to GST alone. A report has shown that the C2A domain is also capable of binding Ca2+, even though, due to incomplete Ca2+ coordination, the C2A domain has a low intrinsic in vitro affinity for Ca2+ [36]. A direct comparison of the binding of the MBP- STNA and MBP-STNB proteins to the GST-C2B and C2A domains indicated that the binding to C2B was 3 to 4-fold greater than the binding to the C2A domain (Figure S1, n = 4, p,0.05). In fact, the level of binding to the C2A domain is similar to the binding of MBP-Stoned proteins to the GST-C2B domain in Ca2+-free buffer (approximately 30% of binding in Ca2+ buffer), and might suggest that the observed binding of STNB and SYT-I in the presence of EGTA represents non-specific binding and that Ca2+ plays an essential role in the binding of the Stoned proteins to C2B SYT-1. We had previously shown that STNA and STNB both bound preferentially to the C2B rather than the C2A domain of SYT-1 [17]. After being bound in the presence of Ca2+, both STNB and STNA can be dissociated from the C2B domain if the complexes PLoS ONE | www.plosone.org 2 June 2012 | Volume 7 | Issue 6 | e38822