2+ Ca Regulates the Drosophila Stoned-A and Stoned-B Proteins Interaction with the C2B Domain of Synaptotagmin-1 Carolina Soekmadji1*, Clement Angkawidjaja2, Leonard E. Kelly1 1 Department of Genetics, The University of Melbourne, Parkville, Victoria, Australia, 2 Department of Material and Life Science, Osaka University, Suita, Osaka, Japan Abstract The dicistronic Drosophila stoned gene is involved in exocytosis and/or endocytosis of synaptic vesicles. Mutations in either stonedA or stonedB cause a severe disruption of neurotransmission in fruit flies. Previous studies have shown that the coiled- coil domain of the Stoned-A and the m-homology domain of the Stoned-B protein can interact with the C2B domain of Synaptotagmin-1. However, very little is known about the mechanism of interaction between the Stoned proteins and the C2B domain of Synaptotagmin-1. Here we report that these interactions are increased in the presence of Ca2+. The Ca2+- dependent interaction between the m-homology domain of Stoned-B and C2B domain of Synaptotagmin-1 is affected by phospholipids. The C-terminal region of the C2B domain, including the tryptophan-containing motif, and the Ca2+ binding loop region that modulate the Ca2+-dependent oligomerization, regulates the binding of the Stoned-A and Stoned-B proteins to the C2B domain. Stoned-B, but not Stoned-A, interacts with the Ca2+-binding loop region of C2B domain. The results indicate that Ca2+-induced self-association of the C2B domain regulates the binding of both Stoned-A and Stoned-B proteins to Synaptotagmin-1. The Stoned proteins may regulate sustainable neurotransmission in vivo by binding to Ca2+- bound Synaptotagmin-1 associated synaptic vesicles. Citation: Soekmadji C, Angkawidjaja C, Kelly LE (2012) Ca2+ Regulates the Drosophila Stoned-A and Stoned-B Proteins Interaction with the C2B Domain of Synaptotagmin-1. PLoS ONE 7(6): e38822. doi:10.1371/journal.pone.0038822 Editor: Karl-Wilhelm Koch, University of Oldenburg, Germany Received February 6, 2012; Accepted May 11, 2012; Published June 12, 2012 Copyright: 2012 Soekmadji et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. * E-mail: carolina.soekmadji@qut.edu.au Introduction Synaptotagmin-1 (SYT-1) is primarily associated with Ca2+- dependent exocytosis of synaptic vesicles (SV). An integral membrane component of SV, SYT-1 is essential for fast Ca2+- dependent release of neurotransmitter in Drosophila, C. elegans, and mouse [1,2,3,4,5,6,7] Structurally synaptotagmins consist of a short luminal domain, a single transmembrane segment, and two tandem C2 domains (C2A and C2B). These C2 domains form Ca2+-dependent complexes with the SNARE proteins and hence trigger exocytosis [8,9,10]. Initial studies using Drosophila have given insight into the essential function of the C2A and C2B domains of SYT-1 [7]. Flies that lack both these domains (AD4 flies) have an equivalent phenotype to SYT-1 flies, as indicated by the absence of fast neurotransmitter release [7]. The C2B deleted-mutant flies (AD1 flies) show a loss of Ca2+ cooperativity for synchronous neurotransmitter release and a defect in endocytosis as indicated by depletion of SV, which suggest that the C2B domain is essential for SV cycling. The AD1 phenotype is thought to be due to the inability of SYT-1 to bind the SNARE complex, and the inability of SYT-1 to oligomerize [7]. As well as being the presumptive Ca2+ sensor for neurotransmitter release, SYT-1 has also been implicated in endocytosis of fused SV. The C2B domain of SYT-1 binds a number of important proteins and moieties associated with exo- and endocytosis, such as syntaxin, SNAP-25, m2 and a-adaptin of AP-2, phospholipids and the Stoned proteins [10,11,12,13,14,15,16,17]. The C2B domain appears to be the target for induction of endocytosis, and it has been shown that it is recognized by components of the AP-2, and hence clathrin-dependent endocytic machinery. This interaction was originally thought to be Ca2+-independent and via the lysine- rich motif [18,19]. Further study has shown the interaction of AP- 2 with SYT-1 to be Ca2+ and phosphatidylserine-dependent and to involve the m2 subunit of AP-2 interacting with the lysine-rich motif of C2B SYT-1 [20]. In Drosophila, while this lysine-rich motif may be important for defining the size of recycled synaptic vesicles and in the priming/ docking step of vesicle exocytosis [21,22], it plays no role in their rates of endocytosis [21]. The lysine-rich motif mutant suffers a defect in the priming/docking step of vesicle exocytosis [22], causing a ,35% reduction in the evoked response, and a two fold increase in spontaneous neurotransmitter release [23]. The corresponding mutation in rat (PC12 cells) has been shown to significantly reduce the extent of SYT-1 internalization (.80%) [24], suggesting that the polyK residues are important for both exocytosis and endocytosis. Studies using a GST tagged C2A-C2B domain of SYT-1, in which the polyK residues are mutated to alanines (A), show abolition of C2B oligomerization [25]. These residues are also shown to be the binding site for the synprint peptide derived from the N and P/Q type Ca2+ channel and AP-2 [19], and further shown to specifically bind to a-adaptin and m2 subunit of AP-2 [15], suggesting that the oligomerization of SYT-1 is important for its function in regulating exo- and endocytosis. PLoS ONE | www.plosone.org 1 June 2012 | Volume 7 | Issue 6 | e38822