- Email: [email protected]
A tale of two C2 domains
2
Update
TRENDS in Neurosciences Vol.26 No.1 January 2003
| Research Focus
A tale of two C2 domains Leo Pallanck Department of Genome Sciences, Box 357730, University of Washington, Seattle, WA 98195, USA
Ca21 influx into the presynaptic terminal triggers fusion of synaptic vesicles with the plasma membrane and release of neurotransmitters. A decade of intensive study of the synaptic vesicle protein synaptotagmin I has led to the general belief that this polypeptide functions as a Ca21 sensor in this process. Previous biochemical work on synaptotagmin I has centered on its two Ca21-binding C2 domains, designated C2A and C2B, which mediate the phospholipid- and effector-proteinbinding interactions believed to be essential to its function. Recent tests of this hypothesis reveal that surprisingly, the C2A-mediated interactions are fully dispensable in vivo. However, at least some of the C2B-mediated interactions appear crucial to synaptotagmin I function. Our current understanding of neurotransmitter release mechanisms derives in large measure from biochemical studies that have used synaptic membranes to identify candidate components of the neurotransmitter release apparatus. This work has culminated in the identification of all major proteins of synaptic vesicles and has facilitated functional inferences based on sequence motifs in these proteins [1]. One of the more interesting molecules identified in this analysis, synaptotagmin I, was found to contain a pair of homologous cytoplasmic domains, designated C2A and C2B, that are themselves homologous to a domain found in isoforms of protein kinase C that bind membranes in response to Ca2þ [2] (Fig. 1). Given that neurotransmitter release is triggered by Ca2þ influx into the presynaptic terminal [3], this observation led to the suggestion that synaptotagmin I, through the activities of its C2 domains, could bind Ca2þ and then proceed to catalyze the membrane-fusion event [2,4]. This early observation captured the interest of many researchers and stimulated a decade of intense effort to explore this model of synaptotagmin I function further. Biochemical characterization of synaptotagmin Subsequent biochemical and structural studies of synaptotagmin I have verified many of the predictions of sequence motifs in this polypeptide. Both of the synaptotagmin I C2 domains bind Ca2þ, with the C2A and C2B domains capable of binding up to three Ca2þ and two Ca2þ, respectively [5,6] (Fig. 1). At Ca2þ concentrations sufficient to induce neurotransmitter release, both of the C2 domains bind negatively charged phospholipids [5] suggesting that one or both of these domains might serve as the Ca2þ sensor that triggers bilayer fusion. Binding of Ca2þ to the Corresponding author: Leo Pallanck ([email protected]).
C2A domain also induces interactions of synaptotagmin I with syntaxin [6 – 8], a member of the SNAP receptor (SNARE) family of membrane proteins that function at a late step (possibly the membrane-fusion event) of all types of vesicle transport [9]. This finding has led to the suggestion that synaptotagmin I, through the activity of the C2A domain, could impart Ca2þ-regulation on the functions of the SNARE proteins. Additional studies indicate that the C2B domain mediates Ca2þ-dependent oligomerization of synaptotagmin I [10,11] and binds a variety of factors implicated in synaptic transmission, including Ca2þ channels [12] and the clathrin adaptor protein AP-2 [13]. The C2B binding interactions suggest • Ca2+-induced phospholipid • binding
• Ca2+-induced phospholipid • binding • Ca2+-induced syntaxin • binding
• Ca2+-induced oligomerization • Ca2+-channel binding • AP-2 binding • SV2 binding • β-SNAP binding • Inositol polyphosphate • binding
C2 A
C2 B N416
N362
N229 S D282 D229
D284
Ca2+ Ca2+ Ca2+ D223 G
D290
N418
D416
D418
D362 Ca2+ D356
Ca2+ D424
N356 TRENDS in Neurosciences
Fig. 1. Structural and functional organization of synaptotagmin I. Synaptotagmin I is an integral membrane protein of synaptic vesicles that has a short intravesicular domain, a membrane-spanning domain and a pair of homologous cytoplasmic domains designated C2A and C2B. Most of the cytoplasmic portion of synaptotagmin consists of the C2 domains. Some of the putative functional roles of the C2A and C2B domains determined from biochemical analyses are listed above each domain. Below each C2 domain are the evolutionarily conserved aspartate (D) residues in Drosophila synaptotagmin I that are believed to coordinate binding to Ca2þ. Blue lines represent specific coordination contacts between aspartate residues and Ca2þ. The green residues shown adjacent to D223 and D282 in the C2A domain represent serine (S) and glycine (G) residues found in the corresponding positions of Drosophila synaptotagmin IV. Arrows indicate amino acids substituted with asparagine (N) by Robinson et al. [20] and Mackler et al. [22] to study the contributions of Ca2þ-binding by these domains to synaptotagmin I function. The D229 residue in the C2A domain is responsible for high-affinity Ca2þ binding to this domain. Studies of the C2B domain by Mackler et al. involved a synaptotagmin I derivative bearing the D416N and D418N substitutions, and a more severely affected D356N and D362N substitution derivative. Abbreviations: AP-2, clathrin adaptor protein 2; SNAP, soluble NSF-attachment protein; SV2, synaptic-vesicle protein 2.
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Update
TRENDS in Neurosciences Vol.26 No.1 January 2003
that synaptotagmin I could also function to localize vesicles near to sites of Ca2þ influx and/or mediate synaptic vesicle recycling. In summary, these biochemical studies strongly supported a role for synaptotagmin I as a Ca2þ sensor in neurotransmitter release, with the C2A domain playing perhaps the major role in this process. In vivo functional analysis of synaptotagmin: a test of the role of the C2A domain To test the validity of models of synaptotagmin function derived from biochemical data, studies of this protein were initiated in a variety of model organisms. Early analysis in the squid system showed that injection of peptides corresponding to several different C-terminal domains of synaptotagmin I inhibited neurotransmitter release, presumably by interfering with the ability of endogenous synaptotagmin to interact with effector proteins [14]. Importantly, these peptides were found to inhibit neurotransmitter release at a step downstream of synaptic vesicle docking, consistent with a late step for synaptotagmin I in Ca2þ-mediated exocytosis. Subsequent genetic studies in worms [15], flies [16,17] and mice [18] also support an essential requirement for synaptotagmin in neurotransmitter release. Although residual neuronal function in several of these knockout animals was initially cited as evidence against synaptotagmin having a central role as the Ca2þ sensor [15,16], it is now clear that synaptotagmin I is just one member of a large family of related factors [19], raising the possibility that residual synaptic transmission in the knockout animals results from functional redundancy. These in vivo analyses of synaptotagmin function have established an important role for this protein in neurotransmitter release. However, these studies have been less than definitive with respect to the role of synaptotagmin as a Ca2þ sensor or regarding the functional significance of particular Ca2þ-mediated synaptotagmin I interactions that have been identified in biochemical studies. However, a recent study in Drosophila has begun to address this latter issue with surprising results [20]. To investigate the importance of Ca2þ binding by the C2A domain of Drosophila synaptotagmin I, Robinson et al. replaced an aspartate residue in C2A that coordinates binding of a high-affinity Ca2þ ion with an asparagine residue incapable of this interaction (Fig. 1). Although the effect of this mutation on Ca2þ binding was not documented in this work, Robinson et al. established that a recombinant C2A domain bearing this alteration is incapable of significant Ca2þ-induced binding of anionic phospholipids or syntaxin at Ca2þ concentrations as high as 1 mM . A cDNA encoding this altered synaptotagmin was then introduced into the Drosophila germline and tested for its ability to rescue the physiological phenotypes resulting from loss of endogenous synaptotagmin I function. Remarkably, this transgene was found to confer substantial rescue of the synaptic transmission defect of synaptotagmin-I-null mutants, indicating that Ca2þ-induced binding of phospholipids and syntaxin by C2A are not essential functions of synaptotagmin I. To substantiate the validity of these findings further, Robinson et al. tested whether transgenic expression of http://tins.trends.com
3
synaptotagmin IV – an isoform of synaptotagmin I lacking several aspartate residues in the C2A domain that are required for efficient Ca2þ binding (Fig. 1) – could also rescue the synaptotagmin-I-null phenotype. Previous work on Drosophila synaptotagmin IV established that the C2A domain of this isoform is incapable of Ca2þ-induced binding of phospholipids or syntaxin, and that overexpression of this protein results in a mild inhibition of neurotransmission. This led to the conclusion that synaptotagmin IV functions as an inhibitor of neurotransmitter release [21]. Nevertheless, Robinson, et al., found that transgenic expression of synaptotagmin IV also rescued the synaptotagmin-I-null physiological phenotypes and conferred a normal Ca2þ-dependency on vesicle release, further demonstrating the dispensable nature of Ca2þ-induced binding of syntaxin and phospholipids conferred by the C2A domain. Subsequent experiments by Robinson et al., to re-examine the previously reported inhibitory effects of synaptotagmin IV overexpression on neurotransmission, failed to replicate these findings. Although this discrepancy requires further explanation, the present results demonstrating rescue of the synaptotagmin-I-mutant phenotypes with a transgene encoding synaptotagmin IV lead to the inescapable conclusion that synaptotagmin IV promotes, rather than inhibits, neurotransmitter release. So, is synaptotagmin I a Ca21 sensor in neurotransmitter release? The results of Robinson et al. raise serious questions about the requirement for Ca2þ-induced binding of anionic phospholipids and syntaxin by the C2A domain. However, this study does not put to rest the question of whether synaptotagmin I functions as a primary Ca2þ sensor in neurotransmitter release. A paper by Mackler et al. in the same issue of Nature describes use of a similar mutational approach in Drosophila but shows, in stark contrast to the results of Robinson et al., that derivatives of synaptotagmin I that bear C2B alterations designed to impair Ca2þ-binding (Fig. 1) are completely unable to rescue the synaptotagmin-I-null physiological phenotypes [22]. In fact, careful examination of the effects of these transgenes revealed that they confer substantial inhibitory effects on the residual neurotransmission present in synaptotagminI-null mutants and significantly reduce the Ca2þ-dependency of release. Similar inhibitory effects were also observed following expression of these transgenes in wild-type flies. These results establish the importance of Ca2þ binding by C2B and, together with the results of Robinson et al., raise the possibility that C2B, and not C2A, might represent the primary Ca2þ-sensing domain required for synaptic vesicle fusion. To explore the mechanism by which these C2B alterations inhibit neurotransmitter release, Mackler et al. tested the effects of the C2B alterations on processes thought to be regulated by this domain, including synaptic vesicle recycling, Ca2þ driven oligomerization and binding to negatively charged phospholipids. These experiments failed to document significant effects of altering the synaptotagmin I C2B domain on synaptic vesicle recycling and revealed only modest effects on Ca2þ-stimulated
Update
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TRENDS in Neurosciences Vol.26 No.1 January 2003
oligomerization. By contrast, the C2B domain alterations had a major effect on Ca2þ-stimulated binding of anionic phospholipids, which roughly paralleled the severity of their effects on neurotransmission, tempting speculation that this activity could represent the primary functional role of the C2B domain. If this conclusion is correct, given the results of Robinson et al. documenting similarly deleterious effects of the C2A mutations on phospholipid binding, it implies that C2B plays the more significant role in this process. Furthermore, this conclusion invites a much more difficult question: what is the precise role of phospholipid binding by C2B? A definitive resolution of this issue might not be forthcoming for quite some time. In summary, after more than a decade of intensive investigation of synaptotagmin function, these two studies in Drosophila make clear that there is still much we need to learn about this molecule if we are to explain fully its role in neurotransmitter release. If the past is any indicator, there will surely be many more surprises (and controversies) in store for us before we are finished with this fascinating protein. Acknowledgements I thank M. Babcock and J. Golby for their critical reading of the manuscript and helpful suggestions. References 1 Sudhof, T.C. et al. (1993) Membrane fusion machinery: insights from synaptic proteins. Cell 75, 1 – 4 2 Perin, M.S. et al. (1990) Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C. Nature 345, 260– 263 3 Katz, B. (1969) The Release of Neural Transmitter Substances, Liverpool University Press 4 Brose, N. et al. (1992) Synaptotagmin: a calcium sensor on the synaptic vesicle surface. Science 256, 1021 – 1025 5 Fernandez, I. et al. (2001) Three-dimensional structure of the synaptotagmin 1 C2B-domain. Synaptotagmin 1 as a phospholipid binding machine. Neuron 32, 1057 – 1069
6 Ubach, J. et al. (1998) Ca2þ binding to synaptotagmin: how many Ca2þ ions bind to the tip of a C2-domain? EMBO J. 17, 3921– 3930 7 Li, C. et al. (1995) Distinct Ca2þ and Sr2þ binding properties of synaptotagmins. Definition of candidate Ca2þ sensors for the fast and slow components of neurotransmitter release. J. Biol. Chem. 270, 24898 – 24902 8 Kee, Y. and Scheller, R.H. (1996) Localization of synaptotagminbinding domains on syntaxin. J. Neurosci. 16, 1975 – 1981 9 Weber, T. et al. (1998) SNAREpins: minimal machinery for membrane fusion. Cell 92, 759 – 772 10 Sugita, S. et al. (1996) Distinct Ca2þ-dependent properties of the first and second C2-domains of synaptotagmin I. J. Biol. Chem. 271, 1262– 1265 11 Chapman, E.R. et al. (1996) A novel function for the second C2 domain of synaptotagmin. Ca2þ-triggered dimerization. J. Biol. Chem. 271, 5844– 5849 12 Sheng, Z.H. et al. (1997) Interaction of the synprint site of N-type Ca2þ channels with the C2B domain of synaptotagmin I. Proc. Natl Acad. Sci. USA 94, 5405– 5410 13 Zhang, J.Z. et al. (1994) Synaptotagmin I is a high affinity receptor for clathrin AP-2: implications for membrane recycling. Cell 78, 751 – 760 14 Bommert, K. et al. (1993) Inhibition of neurotransmitter release by C2-domain peptides implicates synaptotagmin in exocytosis. Nature 363, 163 – 165 15 Nonet, M.L. et al. (1993) Synaptic function is impaired but not eliminated in C. elegans mutants lacking synaptotagmin. Cell 73, 1291– 1305 16 DiAntonio, A. et al. (1993) Synaptic transmission persists in synaptotagmin mutants of Drosophila. Cell 73, 1281 – 1290 17 Littleton, J.T. et al. (1993) Mutational analysis of Drosophila synaptotagmin demonstrates its essential role in Ca2þ activated neurotransmitter release. Cell 74, 1125 – 1134 18 Geppert, M. et al. (1994) Synaptotagmin I: a major Ca2þ sensor for transmitter release at a central synapse. Cell 79, 717 – 727 19 Sudhof, T.C. (2002) Synaptotagmins: why so many? J. Biol. Chem. 277, 7629– 7632 20 Robinson, I.M. et al. (2002) Synaptotagmins I and IV promote transmitter release independently of Ca2þ binding in the C2A domain. Nature 418, 336 – 340 21 Littleton, J.T. et al. (1999) Function modulated by changes in the ratio of synaptotagmin I and IV. Nature 400, 757 – 760 22 Mackler, J.M. et al. (2002) The C2B Ca2þ-binding motif of synaptotagmin is required for synaptic transmission in vivo. Nature 418, 340– 344
Ubiquitin and synaptic dysfunction: ataxic mice highlight new common themes in neurological disease Michael D. Ehlers Departments of Neurobiology, Cell Biology and Pharmacology, Duke University Medical Center, Box 3209, Durham, NC 27710, USA
The gene responsible for the neurological symptoms in ataxia mice has been identified and shown to encode a ubiquitin-specific protease. This study reveals new links between ubiquitination, synapse function and neurological disease. A final commonality in most neurodegenerative diseases, including Alzheimer’s disease (AD), is loss of neurons from crucial brain areas. However, symptoms of illness often Corresponding author: Michael D. Ehlers ([email protected]).
precede detectable neuronal loss, and many neurological syndromes proceed without appreciable cell death or loss of nervous tissue. In such cases, the underlying deficit is believed to be in how the nerve cells themselves function and communicate at synapses. Indeed, aspects of cognitive impairment in both AD and age-related memory decline have been attributed to synaptic dysfunction [1– 10]. However, uncovering the links between disease genes or risk factors, synapse dysfunction and pathological features of neurological disease remains a formidable challenge. Now, a paper in Nature Genetics from the laboratory of Nancy
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Update
TRENDS in Neurosciences Vol.26 No.1 January 2003
| Research Focus
A tale of two C2 domains Leo Pallanck Department of Genome Sciences, Box 357730, University of Washington, Seattle, WA 98195, USA
Ca21 influx into the presynaptic terminal triggers fusion of synaptic vesicles with the plasma membrane and release of neurotransmitters. A decade of intensive study of the synaptic vesicle protein synaptotagmin I has led to the general belief that this polypeptide functions as a Ca21 sensor in this process. Previous biochemical work on synaptotagmin I has centered on its two Ca21-binding C2 domains, designated C2A and C2B, which mediate the phospholipid- and effector-proteinbinding interactions believed to be essential to its function. Recent tests of this hypothesis reveal that surprisingly, the C2A-mediated interactions are fully dispensable in vivo. However, at least some of the C2B-mediated interactions appear crucial to synaptotagmin I function. Our current understanding of neurotransmitter release mechanisms derives in large measure from biochemical studies that have used synaptic membranes to identify candidate components of the neurotransmitter release apparatus. This work has culminated in the identification of all major proteins of synaptic vesicles and has facilitated functional inferences based on sequence motifs in these proteins [1]. One of the more interesting molecules identified in this analysis, synaptotagmin I, was found to contain a pair of homologous cytoplasmic domains, designated C2A and C2B, that are themselves homologous to a domain found in isoforms of protein kinase C that bind membranes in response to Ca2þ [2] (Fig. 1). Given that neurotransmitter release is triggered by Ca2þ influx into the presynaptic terminal [3], this observation led to the suggestion that synaptotagmin I, through the activities of its C2 domains, could bind Ca2þ and then proceed to catalyze the membrane-fusion event [2,4]. This early observation captured the interest of many researchers and stimulated a decade of intense effort to explore this model of synaptotagmin I function further. Biochemical characterization of synaptotagmin Subsequent biochemical and structural studies of synaptotagmin I have verified many of the predictions of sequence motifs in this polypeptide. Both of the synaptotagmin I C2 domains bind Ca2þ, with the C2A and C2B domains capable of binding up to three Ca2þ and two Ca2þ, respectively [5,6] (Fig. 1). At Ca2þ concentrations sufficient to induce neurotransmitter release, both of the C2 domains bind negatively charged phospholipids [5] suggesting that one or both of these domains might serve as the Ca2þ sensor that triggers bilayer fusion. Binding of Ca2þ to the Corresponding author: Leo Pallanck ([email protected]).
C2A domain also induces interactions of synaptotagmin I with syntaxin [6 – 8], a member of the SNAP receptor (SNARE) family of membrane proteins that function at a late step (possibly the membrane-fusion event) of all types of vesicle transport [9]. This finding has led to the suggestion that synaptotagmin I, through the activity of the C2A domain, could impart Ca2þ-regulation on the functions of the SNARE proteins. Additional studies indicate that the C2B domain mediates Ca2þ-dependent oligomerization of synaptotagmin I [10,11] and binds a variety of factors implicated in synaptic transmission, including Ca2þ channels [12] and the clathrin adaptor protein AP-2 [13]. The C2B binding interactions suggest • Ca2+-induced phospholipid • binding
• Ca2+-induced phospholipid • binding • Ca2+-induced syntaxin • binding
• Ca2+-induced oligomerization • Ca2+-channel binding • AP-2 binding • SV2 binding • β-SNAP binding • Inositol polyphosphate • binding
C2 A
C2 B N416
N362
N229 S D282 D229
D284
Ca2+ Ca2+ Ca2+ D223 G
D290
N418
D416
D418
D362 Ca2+ D356
Ca2+ D424
N356 TRENDS in Neurosciences
Fig. 1. Structural and functional organization of synaptotagmin I. Synaptotagmin I is an integral membrane protein of synaptic vesicles that has a short intravesicular domain, a membrane-spanning domain and a pair of homologous cytoplasmic domains designated C2A and C2B. Most of the cytoplasmic portion of synaptotagmin consists of the C2 domains. Some of the putative functional roles of the C2A and C2B domains determined from biochemical analyses are listed above each domain. Below each C2 domain are the evolutionarily conserved aspartate (D) residues in Drosophila synaptotagmin I that are believed to coordinate binding to Ca2þ. Blue lines represent specific coordination contacts between aspartate residues and Ca2þ. The green residues shown adjacent to D223 and D282 in the C2A domain represent serine (S) and glycine (G) residues found in the corresponding positions of Drosophila synaptotagmin IV. Arrows indicate amino acids substituted with asparagine (N) by Robinson et al. [20] and Mackler et al. [22] to study the contributions of Ca2þ-binding by these domains to synaptotagmin I function. The D229 residue in the C2A domain is responsible for high-affinity Ca2þ binding to this domain. Studies of the C2B domain by Mackler et al. involved a synaptotagmin I derivative bearing the D416N and D418N substitutions, and a more severely affected D356N and D362N substitution derivative. Abbreviations: AP-2, clathrin adaptor protein 2; SNAP, soluble NSF-attachment protein; SV2, synaptic-vesicle protein 2.
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Update
TRENDS in Neurosciences Vol.26 No.1 January 2003
that synaptotagmin I could also function to localize vesicles near to sites of Ca2þ influx and/or mediate synaptic vesicle recycling. In summary, these biochemical studies strongly supported a role for synaptotagmin I as a Ca2þ sensor in neurotransmitter release, with the C2A domain playing perhaps the major role in this process. In vivo functional analysis of synaptotagmin: a test of the role of the C2A domain To test the validity of models of synaptotagmin function derived from biochemical data, studies of this protein were initiated in a variety of model organisms. Early analysis in the squid system showed that injection of peptides corresponding to several different C-terminal domains of synaptotagmin I inhibited neurotransmitter release, presumably by interfering with the ability of endogenous synaptotagmin to interact with effector proteins [14]. Importantly, these peptides were found to inhibit neurotransmitter release at a step downstream of synaptic vesicle docking, consistent with a late step for synaptotagmin I in Ca2þ-mediated exocytosis. Subsequent genetic studies in worms [15], flies [16,17] and mice [18] also support an essential requirement for synaptotagmin in neurotransmitter release. Although residual neuronal function in several of these knockout animals was initially cited as evidence against synaptotagmin having a central role as the Ca2þ sensor [15,16], it is now clear that synaptotagmin I is just one member of a large family of related factors [19], raising the possibility that residual synaptic transmission in the knockout animals results from functional redundancy. These in vivo analyses of synaptotagmin function have established an important role for this protein in neurotransmitter release. However, these studies have been less than definitive with respect to the role of synaptotagmin as a Ca2þ sensor or regarding the functional significance of particular Ca2þ-mediated synaptotagmin I interactions that have been identified in biochemical studies. However, a recent study in Drosophila has begun to address this latter issue with surprising results [20]. To investigate the importance of Ca2þ binding by the C2A domain of Drosophila synaptotagmin I, Robinson et al. replaced an aspartate residue in C2A that coordinates binding of a high-affinity Ca2þ ion with an asparagine residue incapable of this interaction (Fig. 1). Although the effect of this mutation on Ca2þ binding was not documented in this work, Robinson et al. established that a recombinant C2A domain bearing this alteration is incapable of significant Ca2þ-induced binding of anionic phospholipids or syntaxin at Ca2þ concentrations as high as 1 mM . A cDNA encoding this altered synaptotagmin was then introduced into the Drosophila germline and tested for its ability to rescue the physiological phenotypes resulting from loss of endogenous synaptotagmin I function. Remarkably, this transgene was found to confer substantial rescue of the synaptic transmission defect of synaptotagmin-I-null mutants, indicating that Ca2þ-induced binding of phospholipids and syntaxin by C2A are not essential functions of synaptotagmin I. To substantiate the validity of these findings further, Robinson et al. tested whether transgenic expression of http://tins.trends.com
3
synaptotagmin IV – an isoform of synaptotagmin I lacking several aspartate residues in the C2A domain that are required for efficient Ca2þ binding (Fig. 1) – could also rescue the synaptotagmin-I-null phenotype. Previous work on Drosophila synaptotagmin IV established that the C2A domain of this isoform is incapable of Ca2þ-induced binding of phospholipids or syntaxin, and that overexpression of this protein results in a mild inhibition of neurotransmission. This led to the conclusion that synaptotagmin IV functions as an inhibitor of neurotransmitter release [21]. Nevertheless, Robinson, et al., found that transgenic expression of synaptotagmin IV also rescued the synaptotagmin-I-null physiological phenotypes and conferred a normal Ca2þ-dependency on vesicle release, further demonstrating the dispensable nature of Ca2þ-induced binding of syntaxin and phospholipids conferred by the C2A domain. Subsequent experiments by Robinson et al., to re-examine the previously reported inhibitory effects of synaptotagmin IV overexpression on neurotransmission, failed to replicate these findings. Although this discrepancy requires further explanation, the present results demonstrating rescue of the synaptotagmin-I-mutant phenotypes with a transgene encoding synaptotagmin IV lead to the inescapable conclusion that synaptotagmin IV promotes, rather than inhibits, neurotransmitter release. So, is synaptotagmin I a Ca21 sensor in neurotransmitter release? The results of Robinson et al. raise serious questions about the requirement for Ca2þ-induced binding of anionic phospholipids and syntaxin by the C2A domain. However, this study does not put to rest the question of whether synaptotagmin I functions as a primary Ca2þ sensor in neurotransmitter release. A paper by Mackler et al. in the same issue of Nature describes use of a similar mutational approach in Drosophila but shows, in stark contrast to the results of Robinson et al., that derivatives of synaptotagmin I that bear C2B alterations designed to impair Ca2þ-binding (Fig. 1) are completely unable to rescue the synaptotagmin-I-null physiological phenotypes [22]. In fact, careful examination of the effects of these transgenes revealed that they confer substantial inhibitory effects on the residual neurotransmission present in synaptotagminI-null mutants and significantly reduce the Ca2þ-dependency of release. Similar inhibitory effects were also observed following expression of these transgenes in wild-type flies. These results establish the importance of Ca2þ binding by C2B and, together with the results of Robinson et al., raise the possibility that C2B, and not C2A, might represent the primary Ca2þ-sensing domain required for synaptic vesicle fusion. To explore the mechanism by which these C2B alterations inhibit neurotransmitter release, Mackler et al. tested the effects of the C2B alterations on processes thought to be regulated by this domain, including synaptic vesicle recycling, Ca2þ driven oligomerization and binding to negatively charged phospholipids. These experiments failed to document significant effects of altering the synaptotagmin I C2B domain on synaptic vesicle recycling and revealed only modest effects on Ca2þ-stimulated
Update
4
TRENDS in Neurosciences Vol.26 No.1 January 2003
oligomerization. By contrast, the C2B domain alterations had a major effect on Ca2þ-stimulated binding of anionic phospholipids, which roughly paralleled the severity of their effects on neurotransmission, tempting speculation that this activity could represent the primary functional role of the C2B domain. If this conclusion is correct, given the results of Robinson et al. documenting similarly deleterious effects of the C2A mutations on phospholipid binding, it implies that C2B plays the more significant role in this process. Furthermore, this conclusion invites a much more difficult question: what is the precise role of phospholipid binding by C2B? A definitive resolution of this issue might not be forthcoming for quite some time. In summary, after more than a decade of intensive investigation of synaptotagmin function, these two studies in Drosophila make clear that there is still much we need to learn about this molecule if we are to explain fully its role in neurotransmitter release. If the past is any indicator, there will surely be many more surprises (and controversies) in store for us before we are finished with this fascinating protein. Acknowledgements I thank M. Babcock and J. Golby for their critical reading of the manuscript and helpful suggestions. References 1 Sudhof, T.C. et al. (1993) Membrane fusion machinery: insights from synaptic proteins. Cell 75, 1 – 4 2 Perin, M.S. et al. (1990) Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C. Nature 345, 260– 263 3 Katz, B. (1969) The Release of Neural Transmitter Substances, Liverpool University Press 4 Brose, N. et al. (1992) Synaptotagmin: a calcium sensor on the synaptic vesicle surface. Science 256, 1021 – 1025 5 Fernandez, I. et al. (2001) Three-dimensional structure of the synaptotagmin 1 C2B-domain. Synaptotagmin 1 as a phospholipid binding machine. Neuron 32, 1057 – 1069
6 Ubach, J. et al. (1998) Ca2þ binding to synaptotagmin: how many Ca2þ ions bind to the tip of a C2-domain? EMBO J. 17, 3921– 3930 7 Li, C. et al. (1995) Distinct Ca2þ and Sr2þ binding properties of synaptotagmins. Definition of candidate Ca2þ sensors for the fast and slow components of neurotransmitter release. J. Biol. Chem. 270, 24898 – 24902 8 Kee, Y. and Scheller, R.H. (1996) Localization of synaptotagminbinding domains on syntaxin. J. Neurosci. 16, 1975 – 1981 9 Weber, T. et al. (1998) SNAREpins: minimal machinery for membrane fusion. Cell 92, 759 – 772 10 Sugita, S. et al. (1996) Distinct Ca2þ-dependent properties of the first and second C2-domains of synaptotagmin I. J. Biol. Chem. 271, 1262– 1265 11 Chapman, E.R. et al. (1996) A novel function for the second C2 domain of synaptotagmin. Ca2þ-triggered dimerization. J. Biol. Chem. 271, 5844– 5849 12 Sheng, Z.H. et al. (1997) Interaction of the synprint site of N-type Ca2þ channels with the C2B domain of synaptotagmin I. Proc. Natl Acad. Sci. USA 94, 5405– 5410 13 Zhang, J.Z. et al. (1994) Synaptotagmin I is a high affinity receptor for clathrin AP-2: implications for membrane recycling. Cell 78, 751 – 760 14 Bommert, K. et al. (1993) Inhibition of neurotransmitter release by C2-domain peptides implicates synaptotagmin in exocytosis. Nature 363, 163 – 165 15 Nonet, M.L. et al. (1993) Synaptic function is impaired but not eliminated in C. elegans mutants lacking synaptotagmin. Cell 73, 1291– 1305 16 DiAntonio, A. et al. (1993) Synaptic transmission persists in synaptotagmin mutants of Drosophila. Cell 73, 1281 – 1290 17 Littleton, J.T. et al. (1993) Mutational analysis of Drosophila synaptotagmin demonstrates its essential role in Ca2þ activated neurotransmitter release. Cell 74, 1125 – 1134 18 Geppert, M. et al. (1994) Synaptotagmin I: a major Ca2þ sensor for transmitter release at a central synapse. Cell 79, 717 – 727 19 Sudhof, T.C. (2002) Synaptotagmins: why so many? J. Biol. Chem. 277, 7629– 7632 20 Robinson, I.M. et al. (2002) Synaptotagmins I and IV promote transmitter release independently of Ca2þ binding in the C2A domain. Nature 418, 336 – 340 21 Littleton, J.T. et al. (1999) Function modulated by changes in the ratio of synaptotagmin I and IV. Nature 400, 757 – 760 22 Mackler, J.M. et al. (2002) The C2B Ca2þ-binding motif of synaptotagmin is required for synaptic transmission in vivo. Nature 418, 340– 344
Ubiquitin and synaptic dysfunction: ataxic mice highlight new common themes in neurological disease Michael D. Ehlers Departments of Neurobiology, Cell Biology and Pharmacology, Duke University Medical Center, Box 3209, Durham, NC 27710, USA
The gene responsible for the neurological symptoms in ataxia mice has been identified and shown to encode a ubiquitin-specific protease. This study reveals new links between ubiquitination, synapse function and neurological disease. A final commonality in most neurodegenerative diseases, including Alzheimer’s disease (AD), is loss of neurons from crucial brain areas. However, symptoms of illness often Corresponding author: Michael D. Ehlers ([email protected]).
precede detectable neuronal loss, and many neurological syndromes proceed without appreciable cell death or loss of nervous tissue. In such cases, the underlying deficit is believed to be in how the nerve cells themselves function and communicate at synapses. Indeed, aspects of cognitive impairment in both AD and age-related memory decline have been attributed to synaptic dysfunction [1– 10]. However, uncovering the links between disease genes or risk factors, synapse dysfunction and pathological features of neurological disease remains a formidable challenge. Now, a paper in Nature Genetics from the laboratory of Nancy
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