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 μ-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 μ-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.
References
[1]
Littleton JT, Stern M, Schulze K, Perin M, Bellen HJ (1993) Mutational analysis of Drosophila Synaptotagmin demonstrates its essential role in Ca2+-activated neurotransmitter release Cell 74: 1125–1134.
[2]
Nonet ML, Grundahl K, Meyer BJ, Rand JB (1993) Synaptic function is impaired but not eliminated in C. elegans mutants lacking synaptotagmin. Cell 73: 1291–1305.
[3]
DiAntonio A, Schwarz TL (1994) The effect on synaptic physiology of synaptotagmin mutations in Drosophila. Neuron 12: 909–920.
[4]
Geppert M, Goda Y, Hammer RE, Li C, Rosahl TW, et al. (1994) Synaptotagmin 1: a major Ca2+ sensor for transmitter release at a central synapse. Cell 79: 717–727.
[5]
Littleton JT, Stern M, Perin M, Bellen HJ (1994) Calcium-dependence of neurotransmitter release and rate of spontaneous vesicle fusions are altered in Drosophila Synaptotagmin mutants Proceedings of the National Academy of Sciences of the United States of America 91: 10888–10892.
[6]
Fernandez-Chacon R, Konigstorfer A, Gerber SH, Garcia J, Matos MF, et al. (2001) Synaptotagmin I functions as a calcium regulator of release probability. Nature 410: 41–49.
[7]
Littleton JT, Bai JH, Vyas B, Desai R, Baltus AE, et al. (2001) synaptotagmin mutants reveal essential functions for the C2B domain in Ca2+-triggered fusion and recycling of synaptic vesicles in vivo. Journal of Neuroscience 21: 1421–1433.
[8]
Li C, Ullrich B, Zhang JZ, Anderson RGW, Brose N, et al. (1995) Ca2+-dependent and Ca2+-independent activities of neural and nonneural synaptotagmins Nature 375: 594–599.
[9]
Davis AF, Bai JH, Fasshauer D, Wolowick MJ, Lewis JL, et al. (1999) Kinetics of synaptotagmin responses to Ca2+ and assembly with the core SNARE complex onto membranes. Neuron 24: 363–376.
[10]
Gerona RRL, Larsen EC, Kowalchyk JA, Martin TFJ (2000) The C terminus of SNAP-25 is essential for Ca2+-dependent binding of synaptotagmin to SNARE complexes. Journal of Biological Chemistry 275: 6328–6336.
[11]
Chapman ER, An S, Edwardson JM, Jahn R (1996) A novel function for the second C2 domain of synaptotagmin – Ca2+-triggered dimerization. Journal of Biological Chemistry 271: 5844–5849.
[12]
Schiavo G, Stenbeck G, Rothman JE, Sollner TH (1997) Binding of the synaptic vesicle v-SNARE, synaptotagmin, to the plasma membrane t-SNARE, SNAP-25, can explain docked vesicles at neurotoxin-treated synapses. Proceedings of the National Academy of Sciences of the United States of America 94: 997–1001.
[13]
Zhang JZ, Davletov BA, Sudhof TC, Anderson RG (1994) Synaptotagmin I is a high affinity receptor for clathrin AP-2: implications for membrane recycling. Cell 78: 751–760.
[14]
Grass I, Thiel S, Honing S, Haucke V (2004) Recognition of a basic AP-2 binding motif within the C2B domain of synaptotagmin is dependent on multimerization. Journal of Biological Chemistry 279: 54872–54880.
[15]
Haucke V, Wenk MR, Chapman ER, Farsad K, De Camilli P (2000) Dual interaction of synaptotagmin with mu 2-and alpha-adaptin facilitates clathrin-coated pit nucleation. Embo Journal 19: 6011–6019.
[16]
Martens S, Kozlov MM, McMahon HT (2007) How synaptotagmin promotes membrane fusion. Science 316: 1205–1208.
[17]
Phillips AM, Smith M, Ramaswami M, Kelly LE (2000) The products of the Drosophila stoned locus interact with synaptic vesicles via synaptotagmin. Journal of Neuroscience 20: 8254–8261.
[18]
Zhang JZ, Davletov BA, Sudhof TC, Anderson RGW (1994) Synaptotagmin-I is a high-affinity receptor for clathrin-AP-2 – Implications for membrane recycling. Cell 78: 751–760.
[19]
Chapman ER, Desai RC, Davis AF, Tornehl CK (1998) Delineation of the oligomerization, AP-2 binding, and synprint binding region of the C2B domain of synaptotagmin. Journal of Biological Chemistry 273: 32966–32972.
[20]
Grass I, Thiel S, Honig S, Haucke V (2004) Recognition of a basic AP-2 binding motif within the C2B domain of Synaptotagmin is dependent on multimerization. Journal of Biological Chemistry 279: 54872–54880.
[21]
Poskanzer KE, Fetter RD, Davis GW (2006) Discrete residues in the C2B domain of synaptotagmin I independently specify endocytic rate and synaptic vesicle size. Neuron 50: 49–62.
[22]
Loewen CA, Lee SM, Shin YK, Reist NE (2006) C2B polylysine motif of synaptotagmin facilitates a Ca2+-independent stage of synaptic vesicle priming in vivo. Molecular Biology of the Cell 17: 5211–5226.
[23]
Mackler JM, Reist NE (2001) Mutations in the second C-2 domain of synaptotagmin disrupt synaptic transmission at Drosophila neuromuscular junctions. Journal of Comparative Neurology 436: 4–16.
[24]
Jarousse N, Kelly RB (2001) Endocytotic mechanism in synapses. Current Opinion in Cell Biology 13: 461–469.
[25]
Wu Y, He Y, Bai J, Ji SR, Tucker WC, et al. (2003) Visualization of synaptotagmin I oligomers assembled onto lipid monolayers. Proc Natl Acad Sci U S A 100: 2082–2087.
[26]
Jarousse N, Wilson JD, Arac D, Rizo J, Kelly RB (2003) Endocytosis of synaptotagmin 1 is mediated by a novel, tryptophan-containing motif. Traffic 4: 468–478.
[27]
Arac D, Chen X, Khant HA, Ubach J, Ludtke SJ, et al. (2006) Close membrane-membrane proximity induced by Ca(2+)-dependent multivalent binding of synaptotagmin-1 to phospholipids. Nat Struct Mol Biol 13: 209–217.
[28]
Vrljic M, Strop P, Hill RC, Hansen KC, Chu S, et al. (2011) Post-translational modifications and lipid binding profile of insect cell-expressed full-length mammalian synaptotagmin 1. Biochemistry 50: 9998–10012.
[29]
Stimson DT, Estes PS, Smith M, Kelly LE, Ramaswami M (1998) A product of the Drosophila stoned locus regulates neurotransmitter release. Journal of Neuroscience 18: 9638–9649.
[30]
Stimson DT, Estes PS, Rao S, Krishnan KS, Kelly LE, et al. (2001) Drosophila stoned proteins regulate the rate and fidelity of synaptic vesicle internalization. Journal of Neuroscience 21: 3034–3044.
[31]
Fergestad T, Broadie K (2001) Interaction of stoned and synaptotagmin in synaptic vesicle endocytosis. Journal of Neuroscience 21: 1218–1227.
[32]
Andrews J, Smith M, Merakovsky J, Coulson M, Hannan F, et al. (1996) The stoned locus of Drosophila melanogaster produces a dicistronic transcript and encodes two distinct polypeptides. Genetics 143: 1699–1711.
[33]
Phillips AM, Ramaswami M, Kelly LE (2010) Stoned. Traffic 11: 16–24.
[34]
Kelly LE, Phillips AM (2005) Molecular and genetic characterization of the interactions between the Drosophila stoned-B protein and DAP-160 (intersectin). Biochemical Journal 388: 195–204.
[35]
Martina JA, Bonangelino CJ, Aguilar RC, Bonifacino JS (2001) Stonin 2: an adaptor-like protein that interacts with components of the endocytic machinery. J Cell Biol 153: 1111–1120.
[36]
Ubach J, Zhang XY, Shao XG, Sudhof TC, Rizo J (1998) Ca2+ binding to synaptotagmin: how many Ca2+ ions bind to the tip of a C-2-domain? EMBO Journal 17: 3921–3930.
[37]
Desai RC, Vyas B, Earles CA, Littleton JT, Kowalchyck JA, et al. (2000) The C2B domain of synaptotagmin is a Ca2+-sensing module essential for exocytosis. Journal of Cell Biology 150: 1125–1135.
[38]
Cheng Y, Sequeira SM, Malinina L, Tereshko V, Sollner TH, et al. (2004) Crystallographic identification of Ca2+ and Sr2+coordination sites in synaptotagmin I C2B domain Protein Science 13: 2665–2672.
[39]
Fernandez I, Arac D, Ubach J, Gerber SH, Shin OH, et al. (2001) Three-dimensional structure of the synaptotagmin 1 C2B-domain: Synaptotagmin 1 as a phospholipid binding machine. Neuron 32: 1057–1069.
[40]
Jung N, Wienisch M, Gu M, Rand JB, Muller SL, et al. (2007) Molecular basis of synaptic vesicle cargo recognition by the endocytic sorting adaptor stonin 2. J Cell Biol 179: 1497–1510.
[41]
Mohrmann R, Matthies HJ, Woodruff E 3rd, Broadie K (2008) Stoned B mediates sorting of integral synaptic vesicle proteins. Neuroscience 153: 1048–1063.
[42]
Petrovich TZ, Merakovsky J, Kelly LE (1993) A genetic analysis of the stoned locus and its interaction with dunce, shibire and supressor of stoned variants of Drosophila melanogaster Genetics 133: 955–965.
[43]
Mackler JM, Drummond JA, Loewen CA, Robinson IM, Reist NE (2002) The C2B Ca2+-binding motif of synaptotagmin is required for synaptic transmission in vivo. Nature 418: 340–344.
[44]
Tamura T, Hou JM, Reist NE, Kidokoro Y (2007) Nerve-evoked synchronous release and high K+-induced quantal events are regulated separately by synaptotagmin I at Drosophila neuromuscular junctions. Journal of Neurophysiology 97: 540–549.
[45]
Fergestad T, Davis W, Broadie K (1999) The Stoned Proteins Regulate Synaptic Vesicle Recycling in the Presynaptic Terminal. Journal of Neuroscience 19: 5847–5860.
[46]
Estes PS, Jackson TC, Stiimson DT, Sanyal S, Kelly LE, et al. (2003) Functional dissection of a eukaryotic dicistronic gene: Transgenic stonedB, but not stonedA, restores normal synaptic properties to Drosophila stoned mutants. Genetics 165: 185–196.
[47]
Ubach J, Lao Y, Fernandez I, Arac D, Sudhof TC, et al. (2001) The C2B domain of synaptotagmin I is a Ca2+-binding module. Biochemistry 40: 5854–5860.
[48]
Phillips JC, Braun R, Wang W, Gumbart J, Tajkhorshid E, et al. (2005) Scalable molecular dynamics with NAMD. J Comput Chem 26: 1781–1802.
[49]
Pedretti A, Villa L, Vistoli G (2004) VEGA–an open platform to develop chemo-bio-informatics applications, using plug-in architecture and script programming. J Comput Aided Mol Des 18: 167–173.
[50]
Humphrey W, Dalke A, Schulten K (1996) VMD: visual molecular dynamics. J Mol Graph 14: 33–38, 27–38.
[51]
Fabiato A (1981) Myoplasmic free calcium concentration reached during the twitch of an intact isolated cardiac cell and during calcium-induced release of calcium from the sarcoplasmic reticulum of a skinned cardiac cell from the adult rat or rabbit ventricle. J Gen Physiol 78: 457–497.
[52]
Zhang XY, Rizo J, Sudhof TC (1998) Mechanism of phospholipid binding by the C(2)A-domain of synaptotagmin I. Biochemistry 37: 12395–12403.
