affected by phospholipids such as PS. We have found that the Ca2+-dependent binding of MBP-STNB to GST-C2B is com- pletely inhibited by the presence of Triton X-100-produced micelles containing phsopholipid mixtures of phosphatidyl etha- nolamine (PE), phosphatidyl choline (PC) and phosphatidyl serine (PS) as compared with the binding in the presence of Ca2+ alone and that the critical phospholipid appears to be PS (*p,0.01, n = 4; Fig 2A). In the case of STNA, phospholipids also appeared to reduce the binding but this reduction is not statistically significant (n = 3; Fig 2B). So, in contrast to the findings with m2 [20], it would appear that lipids might play a role in inhibiting the interaction between STNB and SYT-I. The C-terminal and lysine-rich regions of SYT-1-C2B regulate the binding of STNA and STNB Various motifs of the C2B domain have been implicated in the recycling of SYT-1 from the plasma membrane. It has been reported that, in PC12 cells, the C-terminal WHTL motif acts as an internalization signal to promote endocytosis of SYT-1 [26]. Another motif, the poly-lysine (polyK) region, is a binding site for the m2 of AP-2, as well as a-adaptin [15,19]. To investigate whether any of these endocytic motifs might be responsible for the interaction of C2B with the STNA or STNB proteins, we Interaction between Stoned and Synaptotagmin-1 generated GST-DK, which deletes the polyK containing N- terminal region b-strand 1 to b-strand 4 of C2B. We also generated GST tagged C-terminally truncated versions of C2B domain, GST-DW and GST-DH (Fig. 3). A stop codon was introduced at the loop region, in order to avoid misfolding of the chimeric proteins. The RMSD of the structures during 100 ps MD simulation at 300 K suggested that all the constructs examined have similar structural integrity compared to the full-length C2B (Figure S2). The GST-DW construct deletes both the WHTL motif, while GST-DH includes a deletion of the helix prior to the b-strand containing the WHTL motif, as well as the terminal helix (Figure 3A and 3B). Both helices are absent from C2A domain. The binding of both MBP-STNB and MBP-STNA to GST-DK is much reduced compared to the wild-type GST-C2B fusion (Figure 3C, n = 3, p,0.05). The C2B construct with deletion of the polyK motif has been used to identify a-adaptin, the m2 subunit of AP-2 and synprint binding [15,19]. The deletion of the polyK region could possibly alter the folding behavior of the C2B domain of SYT-1 as it deletes half of the C2B domain. However, even though deletion of this region completely abolished STNA binding the deletion did not abolish the MBP-STNB binding (Figure 3C, n = 3). In contrast, deletions of the C-terminal region produced a considerable increase in the levels of both STNA and Figure 2. Addition of phospholipids mixture reduces the STNB binding to GST-C2B. Binding of MBP STNA binding to GST-C2B is not affected by phospholipids (n = 3). B. Phospholipids mixture containing phosphatidylserine (20% PE: phosphatidylethanolamine; 60% PC: phosphatidylcholine, 20% PS: phosphatidylserine) reduces the binding of MBP-STNB (n = 4) to GST-C2B in comparison with binding in the presence of Ca2+ (*n = 4, p,0.01) or PE:PC+ Ca2+ (**n = 4, p,0.05) to the GST-C2B. ‘‘PE:PC’’ indicates 20%PE:80%PC. ‘‘PE:PC:PS’’ indicates 20%PE:60%PC: 20%PS. The quantified MBP-Stoned proteins are normalized against the amount of eluted GST-C2B. The bound MBP-Stoned proteins are 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 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.g002 PLoS ONE | www.plosone.org 4 June 2012 | Volume 7 | Issue 6 | e38822