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Address correspondence to Edwin R. Chapman, Dept. of Physiology, SMI 129, University of Wisconsin, 1300 University Ave., Madison, WI 53706. Tel.: (608) 263-1762. Fax: (608) 265-5512. E-mail: chapman{at}physiology.wisc.edu
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Abstract |
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Key Words: synaptotagmin; SNARE; membrane fusion; C2 domain; exocytosis
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Introduction |
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The Ca2+-binding synaptic vesicle protein synaptotagmin I (syt) has been proposed to serve as the Ca2+ sensor that regulates secretion (Brose et al., 1992; Geppert et al., 1994; Littleton et al., 1994, 2001; Fernández-Chacón et al., 2001), although this role has been debated (DiAntonio and Schwarz, 1994). Syt I spans the vesicle membrane once and possesses a large cytoplasmic domain largely composed of two C2 domains (Perin et al., 1990). The membrane proximal C2 domain, C2A, penetrates lipid bilayers in response to Ca2+ (Davis et al., 1999), and the membrane distal C2 domain, C2B, mediates Ca2+-triggered oligomerization of syt I (Desai et al., 2000). Interestingly, both C2 domains synergize to directly interact with the target membrane SNAREs (t-SNAREs) syntaxin and SNAP-25, and this interaction can occur at all stages of SNARE complex assembly (for review see Augustine, 2001). These findings raise the possibility that syt could regulate SNARE catalyzed membrane fusion in response to binding Ca2+.
This study investigates the biochemical and functional role of tandem C2 domains for complex formation with t-SNAREs and membranes. Our data suggest that sytSNARE interactions are critical for Ca2+-triggered exocytosis and that the tandem C2 domains of syt exhibit unique properties and possess partially redundant Ca2+ binding sites.
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Results and discussion |
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We next used this assay to examine the function of syt during exocytosis. Previously, we reported that the cytoplasmic domain of recombinant syt I (C2A-C2B) inhibits secretion from cracked PC12 cells (Desai et al., 2000) and proposed that it functions as a dominant negative reagent that blocks homo- and heterooligomerization of syt isoforms in response to Ca2+. This effect is apparent in the RDE secretion assay and serves as a reference point for the new constructs described below (Fig. 2 A). Reverse genetic and biochemical studies by Littleton et al. (2001) revealed that a mutation that disrupts the Ca2+-triggered oligomerization activity of syt I inhibits the ability of docked synaptic vesicles to fuse in response to stimulation.
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These data are consistent with a model in which C2A-C2A and C2A-C2B* inhibit fusion by blocking native sytSNARE interactions. This was tested directly by immunoprecipitating SNARE complexes from brain detergent extracts (BDEs) in the presence of increasing concentrations of C2A-C2A. SNARE complexes were isolated using an antisynaptobrevin antibody. Syt does not bind directly to synaptobrevin thus antisynaptobrevin immunoprecipitations selectively pull-down syt that is bound to SNARE complexes containing both v- and t-SNAREs. As shown in Fig. 2 C, C2A-C2A blocked the binding of native syt to SNARE complexes over precisely the same concentration range that inhibits secretion. Furthermore, maximal inhibition of binding, 50%, coincides with the extent of inhibition observed in the cracked cell assay.
These observations further suggest that inhibition by C2A-C2A is mediated by Ca2+-dependent binding to SNAREs. Although we cannot rule out that C2A-C2A inhibits exocytosis by blocking other unknown interactions, the following evidence argues against this possibility. First, in Fig. 2 D C2A-C2A fails to bind the known syt binding proteins AP-2 (Zhang et al., 1994), SV2 (Schivell et al., 1996), and other copies of syt (Desai et al., 2000); synaptophysin, /ß-SNAP, and synaptobrevin served as negative controls. Second, we have conducted an unbiased search for proteins that bind C2A-C2A but not C2A using affinity chromatography. Both proteins bind several yet-to-be characterized proteins from BDE, but the binding protein profiles for C2A and C2A-C2A were identical (unpublished data; note that C2A-C2B and C2A-C2A cannot efficiently bind SNAREs when fused to glutathione S-transferase and immobilized on beads [Chapman et al., 1996]). Finally, C2A binds with high affinity to acidic phospholipids (40 nM; Davis et al., 1999) as does C2A-C2A. Yet, C2A fails to inhibit secretion. This result is not unexpected; even if C2Amembrane interactions are critical for exocytosis, 10 µM C2A would not be expected to saturate all of the lipid binding sites in the cracked cells. We conclude that the most likely mode of inhibition by C2A-C2A is via blocking native sytSNARE interactions.
If these interactions have a critical role in a late step in secretion, we would expect rapid inhibition regardless of the time at which C2A-C2A was added to the cracked cells. Addition of C2A-C2B* (Fig. 2 E) or C2A-C2A (unpublished data) before, during, or at varying times after the Ca2+ signal resulted in the rapid inhibition of exocytosis, suggesting perturbation of a late step in the fusion reaction.
The ability of tethered C2A domains to bind t-SNAREs further indicates a form of synergy between tandem C2 domains (Chapman et al., 1996; Davis et al., 1999). We further explored this synergy by disrupting the Ca2+ binding ability of each C2 domain in C2A-C2B*; disruption of either C2A or C2B only partially abrogated inhibitory activity in the cracked cell assay (Fig. 3 A). However, simultaneous disruption of both C2 domains strongly reduced inhibitory activity (Fig. 3 A). The effects of these mutations in the release assay were paralleled by their effects on SNAREs binding activity (Fig. 3 B). Titration experiments indicated that mutations within a single C2 domain reduce the affinity for SNAP-25 by approximately twofold (unpublished data) and shift the inhibitory activity of C2A-C2B* by roughly the same factor (Fig. 3 A).
We note that earlier studies demonstrated that neutralization of Ca2+ ligands in C2A blocked Ca2+-triggered t-SNARE and membrane interactions (Davis et al., 1999; Bai et al., 2000). However, previous studies made use of the initial syt I cDNA (Perin et al., 1990) that was shown recently to harbor a mutation (D374) that disrupts the activity of the C2B domain (Osborne et al., 1999; Desai et al., 2000). Because all other known syt sequences harbor a glycine at this position (G374), it is unclear whether the D374 variant is a bona fide version of syt I. In Fig. 3 B, we have confirmed that mutations in the C2A domain of D374-syt I indeed disrupt Ca2+-triggered t-SNARE binding activity.
To test whether the apparent partial redundancy between the Ca2+ ligands in the C2 domains of syt I (G374) occurs with other isoforms, we generated analogous mutations in the C2 domains of syt III; again, complete disruption of Ca2+-triggered t-SNARE binding activity required mutations in both C2 domains (Fig. 3 B, bottom). We saw a similar redundancy when Ca2+-triggered liposome binding activity was measured (Fig. 4, A and C). These findings suggested that like C2A, the C2B domains of syt I (G374) and III may form autonomous Ca2+-triggered membrane binding domains. We tested this possibility and found that the isolated C2B domains fail to bind membranes in response to Ca2+ (Fig. 4 B). Thus, an inactive C2A domain when tethered to an inactive C2B domain gives rise to a protein with Ca2+-triggered lipid binding activity. These data suggest a novel form of cooperation between tandem C2 domains. In one model, C2B can compensate for Ca2+ ligand mutations in C2A such that the lipid binding activity within C2A is restored. Alternatively, an adjacent C2A domain may "activate" cryptic Ca2+-triggered membrane binding activity within C2B. Finally, as a control we confirmed that mutations in the C2A domain of the intact cytoplasmic domain of the D374 version of syt I abolished lipid binding activity (Bai et al., 2000), further establishing that the atypical aspartate residue at position 374 disrupts the function of C2B (Desai et al., 2000).
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The data described here support a functional role for the interaction of syt with SNAREs during Ca2+-triggered exocytosis. We do not yet know which of the numerous isoforms of syt expressed in PC12 cells (Marqueze et al., 2000) regulate secretion. Since all syts that interact with SNAREs are likely to bind via a common mechanism, the C2A-C2A construct would be expected to compete with all endogenous syts in the same way that it blocks syt I binding.
A current goal is to determine the mechanism by which syt influences SNARE catalyzed membrane fusion. Syt binds to the membrane proximal "base" of both t-SNAREs, syntaxin, and SNAP-25 at all stages of SNARE complex assembly (Chapman et al., 1995; Davis et al., 1999; Sutton et al., 1999; Gerona et al., 2000; Littleton et al., 2001). These interactions occur in the absence of Ca2+, but Ca2+ increases their affinity by at least an order of magnitude. We propose that the Ca2+-independent mode of binding poises the release machinery for rapid responses to Ca2+ and that the conformational changes associated with Ca2+-induced increases in affinity have a role in triggering the fusion reaction, potentially by facilitating assembly of SNAREs into an active state that may involve C2B-driven multimerization of SNARE complexes (Desai et al., 2000; Littleton et al., 2001). Recent studies also point to a critical role for the penetration of the C2A domain into membranes (Davis et al., 1999; Bai et al., 2000) in excitation-secretion coupling (Fernández-Chacón et al., 2001). Thus, syt may also operate by altering the physical relationship between the base of the SNARE complex and lipid bilayers (Davis et al., 1999).
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Materials and methods |
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RDE voltammetry release assay
Release profiles were generated by adding 350 µl containing 107 cracked cells to the temperature-controlled incubation chamber (containing an Ag/AgCl reference and platinum auxiliary electrode) set to 37°C. A glassy carbon RDE (Eapp = +500 mV versus Ag/AgCl reference electrode) was placed into the cell suspension and rotated at 3,000 rpm to maintain the cells in rapidly mixing conditions. Once a stable baseline was observed (usually within 2 min), a rapid pulse of Ca2+ was added to the suspension via a Hamilton constant flow rate syringe. Ca2+ releases NE from vesicular stores (
10% of the total NE is released at 80 s in response to 100 µM Ca2+), the released NE is oxidized at the surface of the RDE, and the resulting current is converted to concentration using a standard curve. The current at an RDE is directly proportional to the concentration of the species being oxidized as defined by the Levich equation (Earles and Schenk, 1998). The sensitivity of the RDE assay made it unnecessary to preload PC12 cells with NE.
Analysis of release curves
Release curves were background subtracted to eliminate slight signal drops due to addition of Ca2+. Corrected curves were well fitted by single exponential functions, yielding the time constants and amplitudes of response (Axograph software). The reciprocal of the time constant was plotted versus Ca2+ to determine the [Ca2+]1/2. For inhibition experiments, the percent of inhibition was calculated by comparing initial rates or amplitudes of release profiles generated in the presence and absence of inhibitor. Initial rates were calculated from the slope of a tangent line fitted to the initial linear portion of the release profile. Amplitudes correspond to the cumulative amount of NE released 80 s after the addition of Ca2+.
Recombinant proteins
cDNA encoding rat syt I (D374, Perin et al., 1990; G374, Osborne et al., 1999), rat syt III (Mizuta et al., 1994), human SNAP-25B (Bark and Wilson, 1994), and rat syntaxin 1A (Bennett et al., 1992) were provided by T. Südhof (Howard Hughes Medical Institute, Dallas, TX), G. Schiavo (Imperial Cancer Research Fund, London, UK), S. Seino (Chiba University, Chiba, Japan), R. Scheller (Genentech, Inc., Stanford, CA), and M. Wilson (University of New Mexico, Albuquerque, NM), respectively.
The cytoplasmic (residues 96421), C2A (residues 96265), and C2B (residues 248421) domains of wild-type and mutant versions of syt I (G374 and D374 versions) were prepared as described (Chapman et al., 1995). C2A-C2A subcloned into pGEX 4T-1 via Bam H1-Sal 1 sites is composed of the C2A domain with the linker region (96272) (Sutton et al., 1999) followed by a two amino acid insert (Glu-Leu) and a second C2A domain (142265). The cytoplasmic (residues 290569), the C2A (residues 290420), and the C2B (residues 421569) domains of rat syt III and the indicated point mutations were generated from the sequence reported by Mizuta et al. (1994) and subcloned via EcoR1 and Xho1 sites into pGEX-4T-1. All recombinant constructs were confirmed by DNA sequencing and expressed and purified as described; soluble syt fragments were prepared by thrombin cleavage of glutathione S-transferase fusion proteins (Chapman et al., 1996).
Immunoprecipitation and antibodies
Mouse monoclonal antibodies directed against rat syt I (41.1 and 604.1), syntaxin (HPC-1), synaptobrevin (69.1), SV2 (7G8-IgG), synaptophysin (7.2), and /ß-SNAP (77.1) were provided by S. Engers and R. Jahn (Max-Planck Institute, Göttingen, Germany). The AP-2 antibody (anti
-adaptin; 100/2) was from Sigma-Aldrich. Polyclonal rabbit antibodies generated to the C2B regions of syt I were provided by T.F.J. Martin (University of Wisconsin, Madison, WI); rabbit antibodies directed against the COOH-terminal SNAP25 peptide (residues 195206) were obtained from StressGen Biotechnologies.
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Footnotes |
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* Abbreviations used in this paper: BDE, brain detergent extract; LDCV, large dense core vesicle; NE, norepinephrine; RDE, rotating disc electrode; SNAP-25, synaptosome-associated protein of 25 kD; syt, synaptotagmin I; t-SNARE, target membrane SNARE.
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Acknowledgments |
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This study was supported by grants from the National Institutes of Health GM 56827-01, AHA 9750326N, and the Milwaukee Foundation. E.R. Chapman is a Pew Scholar in the Biomedical Sciences. J. Bai is supported by an American Health Association predoctoral fellowship.
Submitted: 3 May 2001
Accepted: 24 July 2001
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References |
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Augustine, G.J. 2001. How does calcium trigger neurotransmitter release? Curr. Opin. Neuro. 11:320326.
Bai, J., C. Earles, J. Lewis, and E.R. Chapman. 2000. Membrane-embedded synaptotagmin penetrates cis and trans target membranes and clusters via a novel mechanism. J. Biol. Chem. 275:2542725435.
Bark, I.C., and M.C. Wilson. 1994. Human cDNA clones encoding two different isoforms of the nerve terminal protein SNAP-25. Gene. 139:291292.[Medline]
Bennett, M.K., N. Calakos, and R.H. Scheller. 1992. Syntaxina synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones. Science. 257:255259.[Medline]
Brose, N.A., G. Petrenko, T.C. Südhof, and R. Jahn. 1992. Synaptotagmin: a Ca2+ sensor on the synaptic vesicle surface. Science. 256:10211025.[Medline]
Burgoyne, R.D., and A. Morgan. 1998. Calcium sensors in regulated exocytosis. Cell Calcium. 24:367376.[Medline]
Chapman, E.R., P.I. Hanson, S. An, and R. Jahn. 1995. Ca2+ regulates the interaction between synaptotagmin and syntaxin. J. Biol. Chem. 270:2366723671.
Chapman, E.R., S. An, J.M. Edwardson, and R. Jahn. 1996. A novel function for the second C2-domain of synaptotagmin: Ca2+-triggered dimerization. J. Biol. Chem. 271:58445849.
Chapman, E.R., R. Desai, A.F. Davis, and C. Tornhel. 1998. Delineation of the oligomerization, AP-2- and synprint-binding region of the C2B-domain of synaptotagmin. J. Biol. Chem. 273:3296632972.
Chen, Y.A., S.J. Scales, V. Duvvuir, M. Murthy, S.M. Patel, H. Schulman, and R.H. Scheller. 2001. Calcium regulation of exocytosis in PC12 cells. J. Biol. Chem. 276:2668026687.
Davis, A.F., J. Bai, D. Fasshauer, M.J. Wolowick, J.L. Lewis, and E.R. Chapman. 1999. Kinetics of synaptotagmin responses to Ca2+ and assembly with the core SNARE complex onto membranes. Neuron. 24:363376.[Medline]
Desai R., B. Vyas, C. Earles, J.T. Littleton, J. Kowalchyck, T.F.J. Martin, and E.R. Chapman. 2000. The C2B domain of synaptotagmin is a Ca2+ sensing module essential for exocytosis. J. Cell Biol. 150:11251135.
DiAntonio, A., and T.L. Schwarz. 1994. The effects on synaptic physiology of synaptotagmin mutations in Drosophila. Neuron. 12:909920.[Medline]
Earles, C., and J.O. Schenk. 1998. Rotating disk electrode voltammetric measurements of dopamine transporter activity: an analytical evaluation. Anal. Biochem. 264:191198.[Medline]
Elferink, L.A., M.R. Peterson, and R.H. Scheller. 1993. A role for synaptotagmin (p65) in regulated exocytosis. Cell. 72:153159.[Medline]
Fernández-Chacón, R., A. Königstorfer, S.H. Gerber, J. Garcia, M.F. Matos, C.F. Stevens, N. Brose, J. Rizo, C. Rosenmund, and T.C. Südhof. 2001. Synaptotagmin I functions as a calcium regulator of release probability. Nature. 410:4149.[Medline]
Geppert, M., Y. Goda, R.E. Hammer, C. Li, T.W. Rosahl, C. Stevens, and T.C. Südhof. 1994. Synaptotagmin I: a major Ca2+ sensor for transmitter release at a central synapse. Cell. 79:717727.[Medline]
Gerona, R.R.L., E.C. Larsen, J.A. Kowalchyk, and T.F.J. Martin. 2000. The C terminus of SNAP25 is essential for Ca2+-dependent binding of synaptotagmin to SNARE complexes. J. Biol. Chem. 275:63286336.
Klenchin, V.A., J.A. Kowalchyk, and T.F.J. Martin. 1998. Large dense-core vesicle exocytosis in PC12 cells. Methods: a companion to methods in enzymology. Methods. 16:204208.[Medline]
Lee, J., and B.R. Lentz. 1998. Secretory and viral fusion may share mechanistic events with fusion between curved lipid bilayers. Proc. Natl. Acad. Sci. USA. 95:92749279.
Littleton, J.T., M. Stern, M. Perin, and H.J. Bellen. 1994. Calcium dependence of neurotransmitter release and rate of spontaneous vesicle fusions are altered in Drosophila synaptotagmin mutants. Proc. Natl. Acad. Sci. USA. 91:1088810892.
Littleton, J.T., J. Bai, B. Vyas, R. Desai, A.E. Baltus, M.B. Garment, S.D. Carlson, B. Ganetzky, and E.R. Chapman. 2001. Synaptotagmin mutants reveal essential functions for the C2B-domain in Ca2+-triggered fusion and recycling of synaptic vesicles in vivo. J. Neurosci. 21:14211433.
Marqueze, B., F. Berton, and M. Seagar. 2000. Synaptotagmins in membrane traffic: which vesicles do the tagmins tag? Biochimie. 82:409420.[Medline]
Mizuta, M.N., N. Inagaki, Y. Nemoto, S. Matsukura, M. Takahashi, and S. Seino. 1994. Synaptotagmin III is novel isoform of rat synaptotagmin expressed in endocrine and neuronal cells. J. Biol. Chem. 269:1167511678.
Osborne, S.L., J. Herreros, P.I.H. Bastiaens, and G. Schiavo. 1999. Calcium-dependent oligomerization of synaptotagmins I and II. J. Biol. Chem. 274:5966.
Perin, M.S., V.A. Fried, G.A. Mignery, R. Jahn, and T.C. Südhof. 1990. Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C. Nature. 345:260263.[Medline]
Sabatini, B.L., and W.G. Regehr. 1996. Timing of neurotransmission at fast synapses in the mammalian brain. Nature. 384:170172.[Medline]
Schivell, A.E., R.H. Batchelor, and S.M. Bajjalieh. 1996. Isoform-specific, Ca2+-regulated interaction of the synaptic vesicle proteins SV2 and synaptotagmin. J. Biol. Chem. 271:2777027775.
Söllner, T., S.W. Whiteheart, M. Brunner, H. Erdjument-Bromage, S. Geromanos, P. Tempst, and J.E. Rothman. 1993. SNAP receptors implicated in vesicle targeting and fusion. Nature. 362:318324.[Medline]
Sutton, R.B., B.A. Davletov, A.M. Berghuis, T.C. Südhof, and S.R. Sprang. 1995. Structure of the first C2-domain of synaptotagmin 1: a novel Ca2+/phospholipid-binding fold. Cell. 80:929938.[Medline]
Sutton, R.B., D. Fasshauer, R. Jahn, and A.T. Brunger. 1998. Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 Å resolution. Nature. 395:347353.[Medline]
Sutton, R.B., J.A. Ernst, and A.T. Brunger. 1999. Crystal structure of the cytosolic C2A-C2B domains of synaptotagmin III: implications for Ca2+-independent SNARE complex interaction. J. Cell Biol. 147:589598.
Weber, T., B.V. Zemelman, J.A. McNew, B. Westerman, M. Gmachl, F. Parlati, T.H. Söllner, and J.E. Rothman. 1998. SNAREpins: minimal machinery for membrane fusion. Cell. 92:759772.[Medline]
Zhang, J.Z., B.A. Davletov, T.C. Südhof, and R.G.W. Anderson. 1994. Synaptotagmin I is a high affinity receptor for clathrin AP-2: implications for membrane recycling. Cell. 78:751760.[Medline]
Zhang, X.Y., J. Rizo, and T.C. Südhof. 1998. Mechanism of phospholipid binding by the C(2)A-domain of synaptotagmin I. Biochemistry. 37:1239512403.[Medline]