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W1982257516 abstract "The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca2+ sensor for rapid exocytosis. Synaptotagmin spans the vesicle membrane once and possesses a cytoplasmic domain largely comprised of two C2 domains designated C2A and C2B. We have determined how deep the Ca2+-binding loops of Ca2+·C2A penetrate into the lipid bilayer and report mutations in synaptotagmin that can uncouple membrane penetration from Ca2+-triggered interactions with the SNARE complex. To determine whether C2A penetrates into the vesicle (“cis”) or plasma (“trans”) membrane, we reconstituted a fragment of synaptotagmin that includes the membrane-spanning and C2A domain (C2A-TMR) into proteoliposomes. Kinetics experiments revealed that cis interactions are rapid (≤500 μs). Binding in the trans mode was distinguished by the slow diffusion of trans target vesicles. Both modes of binding were observed, indicating that the linker between the membrane anchor and C2A domain functions as a flexible tether. C2A-TMR assembled into oligomers via a novel N-terminal oligomerization domain suggesting that synaptotagmin may form clusters on the surface of synaptic vesicles. This novel mode of clustering may allow for rapid Ca2+-triggered oligomerization of the protein via the membrane distal C2B domain. The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca2+ sensor for rapid exocytosis. Synaptotagmin spans the vesicle membrane once and possesses a cytoplasmic domain largely comprised of two C2 domains designated C2A and C2B. We have determined how deep the Ca2+-binding loops of Ca2+·C2A penetrate into the lipid bilayer and report mutations in synaptotagmin that can uncouple membrane penetration from Ca2+-triggered interactions with the SNARE complex. To determine whether C2A penetrates into the vesicle (“cis”) or plasma (“trans”) membrane, we reconstituted a fragment of synaptotagmin that includes the membrane-spanning and C2A domain (C2A-TMR) into proteoliposomes. Kinetics experiments revealed that cis interactions are rapid (≤500 μs). Binding in the trans mode was distinguished by the slow diffusion of trans target vesicles. Both modes of binding were observed, indicating that the linker between the membrane anchor and C2A domain functions as a flexible tether. C2A-TMR assembled into oligomers via a novel N-terminal oligomerization domain suggesting that synaptotagmin may form clusters on the surface of synaptic vesicles. This novel mode of clustering may allow for rapid Ca2+-triggered oligomerization of the protein via the membrane distal C2B domain. N-terminal C2 domain of synaptotagmin C-terminal C2 domain of synaptotagmin first C2 domain and the transmembrane domain of synaptotagmin cytoplasmic domain of synaptotagmin glutathioneS-transferase polyacrylamide gel electrophoresis soluble NSF attachment protein receptor phosphatidylserine phosphatidylcholine dithiothreitol fluorescence resonance energy transfer synaptosomal-associated protein of 25 kDa synthetic dansyl-phosphatidylethanolamine 7-Br2-PC, 1-palmitoyl-2-stearoyl-(6,7)-dibromo-sn-glycero-3-phosphocholine 12-Br2-PC, 1-palmitoyl-2-stearoyl-(11,12)-dibromo-sn-glycero-3-phosphocholine Ca2+-triggered fusion of synaptic vesicles with presynaptic plasma membrane mediates the release of neurotransmitters from neurons. The release process is extremely fast, occurring on the sub-millisecond time scale (1Llinas R. Steinberg I.Z. Walton K. Biophys. J. 1981; 33: 323-352Abstract Full Text PDF PubMed Scopus (496) Google Scholar, 2Heidelberger R. Heinemann C. Neher E. Matthews G. Nature. 1994; 371: 513-515Crossref PubMed Scopus (621) Google Scholar, 3Sabatini B.L. Regehr W.G. Nature. 1996; 384: 170-172Crossref PubMed Scopus (312) Google Scholar). Synaptotagmin I (hereafter referred to as synaptotagmin) is a Ca2+-binding synaptic vesicle protein that has been proposed to function as a Ca2+ sensor that triggers release in response to Ca2+ influx (4Geppert M. Goda Y. Hammer R.E. Li C. Rosahl T.W. Stevens C.F. Südhof T.C. Cell. 1994; 79: 717-727Abstract Full Text PDF PubMed Scopus (1212) Google Scholar, 5Littleton J.T. Stern M. Perin M. Bellen H.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10888-10892Crossref PubMed Scopus (245) Google Scholar, 6DiAntonio A. Schwarz T.L. Neuron. 1994; 12: 909-920Abstract Full Text PDF PubMed Scopus (213) Google Scholar). Structurally, synaptotagmin spans the vesicle membrane once and has a short intravesicular N-terminal domain and a large C-terminal cytoplasmic region that contains two C2 domains, designated C2A and C2B1 (7Perin M. Fried V.A. Mignery G.A. Jahn R. Südhof T.C. Nature. 1990; 345: 260-263Crossref PubMed Scopus (649) Google Scholar). A charged “linker” segment connects the C2 domains to the transmembrane domain. The structure and biochemical properties of the C2A domain have been studied in detail. This domain forms a compact eight-stranded β-sandwich structure. Three flexible loops that protrude from one end of the domain mediate the binding of two to three Ca2+ ions (8Sutton R.B. Davletov B.A. Berghuis A.M. Südhof T.C. Sprang S.R. Cell. 1995; 80: 929-938Abstract Full Text PDF PubMed Scopus (605) Google Scholar, 9Shao X. Fernandez I. Südhof T.C. Rizo J. Biochemistry. 1998; 37: 16106-16115Crossref PubMed Scopus (203) Google Scholar, 10Ubach J. Zhang X.Y. Shao X.G. Südhof T.C. Rizo J. EMBO J. 1998; 17: 3921-3930Crossref PubMed Scopus (250) Google Scholar). Recent fluorescence and NMR studies demonstrated that Ca2+-binding loops 1 and 3 penetrate into lipid bilayers in response to binding Ca2+ (11Chapman E.R. Davis A.F. J. Biol. Chem. 1998; 273: 13995-14001Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 12Davis A.F. Bai J. Fasshauer D. Wolowick M.J. Lewis J.L. Chapman E.R. Neuron. 1999; 24: 363-376Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 13Chae Y.K. Abildgaard F. Chapman E.R. Markley J.L. J. Biol. Chem. 1998; 273: 25659-25663Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The second C2 domain (C2B) mediates the Ca2+-dependent oligomerization of synaptotagmin (14Chapman E.R. An S. Edwardson J.M. Jahn R. J. Biol. Chem. 1996; 271: 5844-5849Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 15Sugita S. Hata Y. Südhof T.C. J. Biol. Chem. 1996; 271: 1262-1265Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), potentially clustering the cytoplasmic domain into a collar or ring-like structure that may regulate the opening or dilation of the fusion pore (16Lindau M. Almers W. Curr. Opin. Cell Biol. 1995; 7: 509-517Crossref PubMed Scopus (211) Google Scholar). Both C2 domains cooperate to mediate high affinity Ca2+-dependent binding of synaptotagmin to the plasma membrane proteins syntaxin (12Davis A.F. Bai J. Fasshauer D. Wolowick M.J. Lewis J.L. Chapman E.R. Neuron. 1999; 24: 363-376Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 14Chapman E.R. An S. Edwardson J.M. Jahn R. J. Biol. Chem. 1996; 271: 5844-5849Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar) and SNAP-25 (17Schiavo G. Stenbeck G. Rothman J.E. Söllner T.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 997-1001Crossref PubMed Scopus (258) Google Scholar, 18Gerona R.R.L. Larsen E.C. Kowalchyk J.A. Martin T.F.J. J. Biol. Chem. 2000; 275: 6328-6336Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Syntaxin and SNAP-25 form a high affinity ternary complex with the synaptic vesicle protein synaptobrevin (19Söllner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2616) Google Scholar, 20Hayashi T. McMahon H. Yamasaki S. Binz T. Hata Y. Südhof T.C. Niemann H. EMBO J. 1994; 13: 5051-5061Crossref PubMed Scopus (661) Google Scholar). This heterotrimer, designated the SNARE complex, has been proposed to function as the core of a conserved membrane fusion “machine” (19Söllner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2616) Google Scholar, 21Weber T. Zemelman B.V. McNew J.A. Westerman B. Gmachl M. Parlati F. Söllner T.H. Rothman J.E. Cell. 1998; 92: 759-772Abstract Full Text Full Text PDF PubMed Scopus (2010) Google Scholar). Synaptotagmin can bind the assembled SNARE complex and penetrate into membranes simultaneously (12Davis A.F. Bai J. Fasshauer D. Wolowick M.J. Lewis J.L. Chapman E.R. Neuron. 1999; 24: 363-376Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). These interactions occur at Ca2+concentrations capable of eliciting exocytosis (2Heidelberger R. Heinemann C. Neher E. Matthews G. Nature. 1994; 371: 513-515Crossref PubMed Scopus (621) Google Scholar) and therefore may couple Ca2+ influx to fusion. These findings prompt a model in which the interaction of the C2A domain with membranes serves to alter the physical relationship between SNAREs and the lipid bilayer. This model prompts the question as to whether Ca2+triggers the interaction of the C2A domain of synaptotagmin with the synaptic vesicle membrane (a “cis” interaction) or whether Ca2+·C2A interacts with the presynaptic plasma membrane (a “trans” interaction). To address this issue we have reconstituted an N-terminal fragment of synaptotagmin that encodes the luminal, transmembrane, linker, and C2A domain of the protein into proteoliposomes. The C2A domain was modified to contain a single fluorescent reporter group, a tryptophan (Trp), which could be used to monitor the penetration of C2A into membranes in real-time (11, 12), and a kinetic approach was used to discern betweencis and trans interactions. In the course of these experiments we observed a novel mode of synaptotagmin oligomerization mediated via the N-terminal region of the protein. Furthermore, we refined the geometry of C2A·membrane interactions by measuring the depth of penetration of the Ca2+-binding loops of C2A. Finally, we observed that Ca2+-triggered synaptotagmin·lipid and synaptotagmin·SNARE interactions can be uncoupled via mutagenesis of the C2A domain making it possible to address discrete synaptotagmin interactions via genetic approaches. cDNAs encoding rat synaptotagmin I (7Perin M. Fried V.A. Mignery G.A. Jahn R. Südhof T.C. Nature. 1990; 345: 260-263Crossref PubMed Scopus (649) Google Scholar), SNAP-25A (22Blasi J. Chapman E.R. Link E. Binz T. Yamasaki S. De Camilli P. Südhof T.C. Niemann H. Jahn R. Nature. 1993; 365: 160-163Crossref PubMed Scopus (1038) Google Scholar), and synaptobrevin II (23Elferink L.A. Trimble W.S. Scheller R.H. J. Biol. Chem. 1989; 264: 11061-11064Abstract Full Text PDF PubMed Google Scholar) were kindly provided by T. C. Südhof (Dallas, TX) and R. Scheller (Stanford, CA). The cytoplasmic domain of synaptotagmin (designated C2AB; amino acids 96–421), C2A domain (residues 96–265), and C2B domain (residues 248–421) were expressed in Escherichia coli as GST fusion proteins, immobilized and purified using glutathione-Sepharose beads (Amersham Pharmacia Biotech) as described (14Chapman E.R. An S. Edwardson J.M. Jahn R. J. Biol. Chem. 1996; 271: 5844-5849Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). In addition, point mutant versions of GST-C2AB (D172N, D178N, D230S, D232N, D238N, D230N/D232N, D309N/D363N/D365N/D371N), were also prepared. All mutagenesis was carried out as described (24Chapman E.R. Jahn R. J. Biol. Chem. 1994; 269: 5735-5741Abstract Full Text PDF PubMed Google Scholar). Full-length rat synaptobrevin II was expressed and purified as described (25Edelmann L. Hanson P. Chapman E.R. Jahn R. EMBO J. 1995; 14: 224-231Crossref PubMed Scopus (388) Google Scholar). Midi SNARE complexes, composed of residues 180–262 of syntaxin 1A, full-length SNAP-25A, and residues 1–96 of synaptobrevin II, were prepared as described (12Davis A.F. Bai J. Fasshauer D. Wolowick M.J. Lewis J.L. Chapman E.R. Neuron. 1999; 24: 363-376Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). C2A-TMRWT encodes residues 1–265 of synaptotagmin I and contains two naturally occurring Trp residues at positions 58 and 259. A mutant version of this fragment, C2A-TMRTrp, was prepared with W58F and W259F substitution mutations to remove the naturally occurring Trp residues. A third mutation, F234W, was introduced such that this construct contains a single Trp, in Ca2+ and lipid-binding loop 3 (11). Recombinant C2A-TMRWT and C2A-TMRTrp were generated as GST fusion proteins by subcloning into pGEX-2T (Amersham Pharmacia Biotech). Proteins were expressed and purified as described above. Soluble C2A-TMRTrp, for fluorescence studies and liposome reconstitution experiments, was expressed using a pTrcHis A vector (InVitrogen) as described (14Chapman E.R. An S. Edwardson J.M. Jahn R. J. Biol. Chem. 1996; 271: 5844-5849Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar), resulting in a fusion protein that contains a His6-/T7-tag at the N terminus. This construct was purified using Ni-NTA-agarose (Qiagen) as described (14Chapman E.R. An S. Edwardson J.M. Jahn R. J. Biol. Chem. 1996; 271: 5844-5849Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). After purification, C2A-TMRTrp was dialyzed against 4 liters of HEPES buffer I (50 mm HEPES-NaOH, pH 7.4, 0.1 mNaCl, and 1% sodium cholate) overnight. For acrylamide and brominated-lipid quenching experiments, soluble C2A, with either a loop 1 Trp reporter (Trp-173) or a loop 3 Trp reporter (Trp-234) were prepared as described (12Davis A.F. Bai J. Fasshauer D. Wolowick M.J. Lewis J.L. Chapman E.R. Neuron. 1999; 24: 363-376Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Brain-derived phosphatidylserine (PS), phosphatidylcholine (PC), synthetic dansyl-phosphatidylethanolamine (dansyl-PE), 1-palmitoyl-2-stearoyl-(6,7)-dibromo-sn-glycero-3-phosphocholine (6,7-Br2-PC) and 1-palmitoyl-2-stearoyl-(11,12)-dibromo-sn-glycero-3-phosphocholine (11,12-Br2-PC) were obtained from Avanti Polar Lipids (Alabaster, AL). To prepare proteoliposomes, lipids were dried and dissolved in HEPES buffer I containing 2 μm protein. Proteoliposomes were formed by dialyzing the lipid/detergent/protein mixture against 4 liters of HEPES buffer II (50 mmHEPES-NaOH, pH 7.4, 0.1 m NaCl) overnight at 4 °C. For fluorescence studies, large (∼100 nm) unilamellar liposomes (26MacDonald R.C. MacDonald R.I. Menco B.P.M. Takeshita K. Subbarao N.K. Hu L.R. Biochim. Biophys. Acta. 1991; 1061: 297-303Crossref PubMed Scopus (1382) Google Scholar) were prepared using an Avanti Polar Lipids extruder according to the manufacturer's instructions. l-3-Phosphatidyl[N-methyl-3H]choline-1,2-dipalmitoyl ([3H]PC) was purchased from Amersham Pharmacia Biotech.3H-Labeled 25% PS/75% PC liposome binding assays were carried out as described (11, 27). In all experiments, error bars represent the standard deviations from triplicate determinations. For Ca2+titration experiments, [Ca2+]free was determined using an MI-600 Ca2+ electrode and an MI-402 microreference electrode (Microelectrode Inc., Bedford, NH) and World Precision Instruments Inc. (Sarasota, FL) Ca2+ standards (pCa2+ range of 1–8). Ca2+ concentrations below 100 μm were buffered using 2 mmEGTA. Proteoliposomes (75 μl aliquots) were collected by centrifugation at 100,000 ×g for 30 min in an Airfuge (Beckman). Pelleted proteoliposomes were resuspended in the indicated buffers and incubated for 20 min at room temperature, and again subjected to centrifugation. 8% of the supernatants and pellets were subjected to SDS-PAGE and immunoblot analysis using anti-synaptotagmin monoclonal antibodies (41.1). Mouse monoclonal antibodies directed against synaptobrevin (69.1) and synaptotagmin (41.1) were kindly provided by R. Jahn (Göttingen, Germany). All immunoprecipitation and bead-binding experiments were carried out at 4 °C. Immunoprecipitation of recombinant SNARE complexes was carried out as described (12Davis A.F. Bai J. Fasshauer D. Wolowick M.J. Lewis J.L. Chapman E.R. Neuron. 1999; 24: 363-376Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Briefly, SNARE complexes were incubated with recombinant synaptotagmin in Tris-buffered saline (TBS; 20 mm Tris, pH 7.4, 150 mm NaCl) plus 0.5% Triton X-100 in the presence of 2 mm EGTA or 1 mm Ca2+ for 2 h. Synaptobrevin was immunoprecipitated by incubating the samples with 69.1 (1.5 μl) ascites for 2 h and 12 μl of protein G-Sepharose Fast-Flow (Amersham Pharmacia Biotech) for 1 h. The immunoprecipitates were washed three times and analyzed by SDS-PAGE and staining with Coomassie blue. As a control for nonspecific precipitation of synaptotagmin, samples were also prepared lacking SNAREs. In each case, the immunoprecipitating antibodies did not bind synaptotagmin, and, under the conditions of the binding assays, synaptotagmin did not precipitate in the absence of SNAREs. Thus, for experiments shown in Fig. 1, samples lacking SNARE complexes (−) also lacked immunoprecipitating antibodies. Co-immunoprecipitation of synaptotagmin was quantified using a Bio-Rad GS-670 imaging densitometer. To avoid increases in synaptotagmin co-immunoprecipitation due to Ca2+-triggered oligomerization, a nonoligomerizing version of synaptotagmin I (7Perin M. Fried V.A. Mignery G.A. Jahn R. Südhof T.C. Nature. 1990; 345: 260-263Crossref PubMed Scopus (649) Google Scholar, 12Davis A.F. Bai J. Fasshauer D. Wolowick M.J. Lewis J.L. Chapman E.R. Neuron. 1999; 24: 363-376Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar) was used in all co-immunoprecipitation experiments. 20 μg of GST, GST-C2A-TMR (amino acids 1–265), GST-C2A (), and GST-synaptobrevin (full-length) immobilized on beads was incubated with 1 μm soluble His6-C2A-TMR (amino acids 1–265) for 4 h in 150 μl of HEPES II buffer in either 2 mm EGTA or 1 mm Ca2+. Beads were washed three times in binding buffer plus 2 mm EGTA or 1 mm Ca2+. 7% of the samples were subjected to SDS-PAGE and immunoblot analysis using monoclonal Penta-His antibodies (Qiagen). Immunoreactive bands were visualized using enhanced chemiluminescence. Steady-state fluorescence measurements were made at 24 °C using a PTI QM-1 fluorometer and Felix software, samples were mixed using a castle-style stir bar. Proteoliposome suspensions containing 2 μm protein and 0.75 mm lipid were mixed with protein free liposomes and excited at 285 nm with 2-nm resolution. For fluorescence resonance energy transfer (FRET) experiments, protein free liposomes contained 5% dansyl-PE, 25% PS, and 70% PC. FRET was monitored by quenching of the Trp donor fluorescence by the dansyl acceptor. Stopped-flow rapid mixing experiments were carried out at 24 °C using an Applied Photophysics SX.18MV stopped-flow spectrometer. Samples were excited at 285 nm, and emitted light was collected using a 326.1-nm bandpass filter. Acrylamide quenching measurements were carried out using a 2 m stock solution of acrylamide. The quencher was added to 7.5 μm C2A-Trp reporter (Trp-173 in loop 1 and Trp-234 in loop 3, respectively) in the presence of either 2 mm EGTA, 1 mmCa2+ or 1 mm Ca2+ plus liposomes (2 mm lipid; 75%PC/25%PS) as indicated. The emission spectra were corrected for blank, dilution, and instrument response and integrated from 300–400 nm to yield the fluorescence intensity. The degree of quenching was analyzed according to the Stern-Volmer equation (Equation 1): Fo/F=1+KSV[Q]Equation 1 where F o and F are the fluorescence intensities in the absence and presence of acrylamide, respectively, K SV is the Stern-Volmer constant for collisional quenching, and [Q] is the concentration of the quencher. The equation predicts a linear plot ofF o/F versus[Q] for a homogeneous solution (28Birks J.B. Photophysics of Aromatic Molecules. Wiley-Interscience, New York1970Google Scholar). The depth measurement was performed according to parallax analysis (29Abrams F.S. London E. Biochemistry. 1992; 31: 5312-5322Crossref PubMed Scopus (104) Google Scholar, 30Chattopadhyay A. London E. Biochemistry. 1987; 26: 39-45Crossref PubMed Scopus (595) Google Scholar, 31Ren J. Lew S. Wang Z. London E. Biochemistry. 1997; 36: 10213-10220Crossref PubMed Scopus (186) Google Scholar). Specifically, the distance of the tryptophan residue from the bilayer center (Z CF) is given by Equation2:ZCF=LC1+[−ln(F1/F2)/πC−L2]/2LEquation 2 where L C1 represents the distance from the bilayer center to the shallow quencher (11 Å for 6,7-Br2-PC), C is the mole fraction of the quencher divided by the lipid area (70 Å2),F 1 and F 2 are the relative fluorescence intensities of the shallow (6,7-Br2-PC) and deep quenchers (11,12-Br2-PC), respectively, and L is the difference in the depth of the two quenchers (0.9 Å per CH2 or CBr2group). For these brominated lipids the thickness of the hydrophobic region is ∼29 Å (32McIntosh T.J. Holloway P.W. Biochemistry. 1987; 26: 1783-1788Crossref PubMed Scopus (165) Google Scholar). Our first goals were to confirm whether the C2A domain of synaptotagmin possesses all of the PS/PC binding activity of the intact cytoplasmic domain of the protein (24Chapman E.R. Jahn R. J. Biol. Chem. 1994; 269: 5735-5741Abstract Full Text PDF PubMed Google Scholar, 27Davletov B.A. Südhof T.C. J. Biol. Chem. 1993; 268: 26386-26390Abstract Full Text PDF PubMed Google Scholar, 33Chapman E.R. Hanson P.I. An S. Jahn R. J. Biol. Chem. 1995; 270: 23667-23671Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar, 34Schiavo G. Gu Q.M. Prestwich G.D. Söllner T.H. Rothman J.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13327-13332Crossref PubMed Scopus (261) Google Scholar) and to compare the roles of the Ca2+ ligands in C2A and C2B for mediating Ca2+-triggered interactions with membranes and the SNARE complex. Structural studies indicate that five acidic amino acid residues (Asp-172, -178, -230, -232, and -238) serve as critical Ca2+ ligands within C2A (8Sutton R.B. Davletov B.A. Berghuis A.M. Südhof T.C. Sprang S.R. Cell. 1995; 80: 929-938Abstract Full Text PDF PubMed Scopus (605) Google Scholar). These ligands are precisely conserved in the C2B domain (7Perin M. Fried V.A. Mignery G.A. Jahn R. Südhof T.C. Nature. 1990; 345: 260-263Crossref PubMed Scopus (649) Google Scholar). As shown in Fig.1 A, neutralization of any of the acidic Ca2+ ligands within C2A, by substitution with Asn or Ser residues, abolished Ca2+-triggered PS/PC liposome binding activity of the intact cytoplasmic domain of synaptotagmin. These data are consistent with studies of the isolated C2A domain (27Davletov B.A. Südhof T.C. J. Biol. Chem. 1993; 268: 26386-26390Abstract Full Text PDF PubMed Google Scholar, 35Zhang X. Rizo J. Südhof T.C. Biochemistry. 1998; 57: 12395-12403Crossref Scopus (166) Google Scholar). In contrast, neutralization of four of the five putative Ca2+ ligands in the C2B domain (D309N/D363N/D365N/D371N) had little effect on Ca2+-triggered PS/PC-binding activity (Fig. 1 A). These data indicate that Ca2+-triggered PS/PC binding activity is regulated by Ca2+ ligands within C2A. A previous report indicated that Asp residue 178 was not essential for Ca2+-triggered binding of the cytoplasmic domain of synaptotagmin to syntaxin (33Chapman E.R. Hanson P.I. An S. Jahn R. J. Biol. Chem. 1995; 270: 23667-23671Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar), suggesting that Ca2+-triggered interactions with lipids and with SNAREs can be uncoupled. However, other reports indicated that the interaction of the isolated C2A domain of synaptotagmin with syntaxin was abolished by neutralization of Asp-178 (36Li C. Ullrich B. Zhang J.Z. Anderson R.G.W. Brose N. Südhof T.C. Nature. 1995; 375: 594-599Crossref PubMed Scopus (541) Google Scholar). We have re-examined this issue by assaying the ability of wild type and mutant forms of the cytoplasmic domain of synaptotagmin to assemble onto the core of the ternary SNARE complex composed of syntaxin, SNAP-25, and synaptobrevin. The fully assembled SDS-resistant SNARE complex (20Hayashi T. McMahon H. Yamasaki S. Binz T. Hata Y. Südhof T.C. Niemann H. EMBO J. 1994; 13: 5051-5061Crossref PubMed Scopus (661) Google Scholar) is shown in Fig.1 B (right upper panel). Because both C2 domains of synaptotagmin are critical for high affinity interactions with SNAREs (12Davis A.F. Bai J. Fasshauer D. Wolowick M.J. Lewis J.L. Chapman E.R. Neuron. 1999; 24: 363-376Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 14Chapman E.R. An S. Edwardson J.M. Jahn R. J. Biol. Chem. 1996; 271: 5844-5849Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 18Gerona R.R.L. Larsen E.C. Kowalchyk J.A. Martin T.F.J. J. Biol. Chem. 2000; 275: 6328-6336Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar), we analyzed three synaptotagmin mutants. Two mutants possessed mutations in the Ca2+ ligands within the C2A domain (D178N and D230,232N) and in the third mutant four of the five conserved Ca2+ ligands within the C2B domain were neutralized (D309N/D363N/D365N/D371N). Each mutant was assayed for co-immunoprecipitation with assembled SNARE complexes using anti-synaptobrevin antibodies in the presence of EGTA or Ca2+. As shown in Fig. 1 B (left panel), wild type and D309N/D363N/D365N/D371N mutant synaptotagmin bound to the SNARE complex in a Ca2+-dependent manner. Consistent with previous reports of synaptotagmin·syntaxin interactions (33Chapman E.R. Hanson P.I. An S. Jahn R. J. Biol. Chem. 1995; 270: 23667-23671Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar), the D178N mutant also bound to the SNARE complex in response to Ca2+. However, 50% of the Ca2+effect was inhibited by this mutation. In contrast, the D230,232N mutant did not exhibit any Ca2+-promoted SNARE complex binding activity but did retain the Ca2+-independent component of binding activity. These data are plotted in Fig.1 B (right lower panel) and demonstrate that, analogous to the binding of anionic phospholipids, all of the Ca2+ ligands that regulate binding to the SNARE complex lie within the C2A domain. However, the D178N mutation can uncouple Ca2+-triggered synaptotagmin·lipid interactions from Ca2+-triggered synaptotagmin·SNARE complex interactions, indicating that these interactions have distinct structural requirements. To formally confirm that C2A mediates all of the anionic phospholipid binding activity of synaptotagmin, equimolar amounts of the cytoplasmic domain (designated C2AB), as well as the isolated C2A and C2B domains, were immobilized as GST fusion proteins and assayed for radiolabeled liposome binding activity in the presence and absence of Ca2+. As shown in Fig.2 A, C2A bound liposomes to the same extent as did C2AB; C2B did not bind liposomes in response to Ca2+. Having established that all of the anionic phospholipid binding activity of synaptotagmin is mediated by the C2A domain, we next sought to generate an optical reporter to follow C2A·membrane interactions using synaptotagmin that has been reconstituted into proteoliposomes via its N-terminal transmembrane segment. The goal of these experiments is to determine whether C2A interacts with the same membrane in which synaptotagmin is embedded (a “cis” interaction) or whether C2A interacts with protein free target vesicles (a “trans” interaction). We therefore prepared a C2A domain that includes the transmembrane segment of synaptotagmin and encompasses amino acids 1–265, where residues 58–79 correspond to the transmembrane anchor and residues 140–265 form the C2A domain. Two versions of this domain were prepared, a wild type (C2A-TMRWT) and mutant form (C2A-TMRTrp) in which all native Trp residues were replaced with phenylalanines, and phenylalanine 234, within Ca2+- and lipid-binding loop 3, was substituted with a tryptophan residue. Thus, C2A-TMRTrpcontains a single Trp reporter, which we have shown in a previous study penetrates into lipid bilayers in response to binding Ca2+(11, 12). Membrane penetration results in an increase in fluorescence intensity and a blue shift in the emission spectrum of the Trp reporter, thus providing a convenient means to monitor Ca2+-triggered C2A·lipid interactions in real-time. C2A-TMR and C2A-TMRTrp bound liposomes to the same extent as the isolated C2A domain of synaptotagmin (Fig. 2A). Furthermore, the Ca2+ sensitivities of C2A, C2A-TMR, and C2A-TMRTrp for binding liposomes were identical (Fig.2 B; [Ca2+]1/2 ∼ 20–21 μm; Hill coefficient 1.9–2.2). These data indicate that C2A-TMRTrp is fully active and is suitable for use in reconstitution experiments, as described below. To determine whether C2A docks onto the same membrane in which synaptotagmin is embedded (i.e. the synaptic vesicle membrane; designated here as a cis interaction) or whether synaptotagmin docks onto a distinct target membrane (i.e. the presynaptic plasma membrane; designated here as atrans interaction) C2A-TMRTrp was generated as a His6-tagged protein and reconstituted into proteoliposomes as described under “Experimental Procedures.” The degree of successful reconstitution was assayed by sedimentation of C2A-TMRTrp proteoliposomes under various conditions. As shown in Fig. 3 A, in HEPES buffer II, all of the C2A-TMRTrp co-sedimented with proteoliposomes. Addition of 1 m salt, a high pH buffer (pH 11) or 5 m urea failed to solubilize the protein, indicating that C2A-TMRTrp was efficiently embedded into proteoliposomes. Surprisingly, 2% Triton solubilized only 50% of the C2A-TMRTrp. Addition of a chaotropic agent (5 murea) or a reducing agent (10 mm DTT) to the 2% Triton buffer resulted in complete solubilization. These data indicate that a secondary process contributes to the sedimentation of C2A-TMRTrp in the co-sedimentation assay. Therefore, before carrying out the cis/trans analysis described further below, we" @default.
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W1982257516 title "Membrane-embedded Synaptotagmin Penetrates cis ortrans Target Membranes and Clusters via a Novel Mechanism" @default.
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W1982257516 doi "https://doi.org/10.1074/jbc.m906729199" @default.
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