The Evolutionary Pressure to Inactivate
A SUBCLASS OF SYNAPTOTAGMINS WITH AN AMINO ACID SUBSTITUTION THAT ABOLISHES Ca2+ BINDING*

(Received for publication, December 4, 1996, and in revised form, February 24, 1997)

Christine von Poser Dagger §, Konstantin Ichtchenko Dagger , Xuguang Shao , Josep Rizo and Thomas C. Südhof Dagger par **

From the Departments of Dagger  Molecular Genetics,  Pharmacology and Biochemistry, and par  Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Synaptotagmin I is a Ca2+-binding protein of synaptic vesicles that serves as a Ca2+ sensor for neurotransmitter release and was the first member found of a large family of trafficking proteins. We have now identified a novel synaptotagmin, synaptotagmin XI, that is highly expressed in brain and at lower levels in other tissues. Like other synaptotagmins, synaptotagmin XI has a single transmembrane region and two cytoplasmic C2-domains but is most closely related to synaptotagmin IV with which it forms a new subclass of synaptotagmins. The first C2-domain of synaptotagmin I (the C2A-domain) binds phospholipids as a function of Ca2+ and contains a Ca2+-binding site, the C2-motif, that binds at least two Ca2+ ions via five aspartate residues and is conserved in most C2-domains (Shao, X., Davletov, B., Sutton, B., Südhof, T. C., Rizo, J. R. (1996) Science 273, 248-253). In the C2A-domains of synaptotagmins IV and XI, however, one of the five Ca2+-binding aspartates in the C2-motif is substituted for a serine, suggesting that these C2-domains do not bind Ca2+. To test this, we produced recombinant C2A-domains from synaptotagmins IV and XI with either wild type serine or mutant aspartate in the C2-motif. Circular dichroism showed that Ca2+ stabilizes both mutant but not wild type C2-domains against temperature-induced denaturation, indicating that the mutations restore Ca2+-binding to the wild type C2-domains. Furthermore, wild type C2A-domains of synaptotagmins IV and XI exhibited no Ca2+-dependent phospholipid binding, whereas mutant C2A-domains bound phospholipids as a function of Ca2+ similarly to wild type synaptotagmin I. These experiments suggest that a class of synaptotagmins was selected during evolution in which the Ca2+-binding site of the C2A-domain was inactivated by a single point mutation. Thus, synaptotagmins must have Ca2+-independent functions as well as Ca2+-dependent functions that are selectively maintained in distinct members of this gene family.


INTRODUCTION

Synaptotagmins represent a large protein family with at least ten genes that probably function in membrane traffic (1-9). All synaptotagmins are characterized by a single N-terminal transmembrane region and two cytoplasmic C2-domains followed by a short conserved C terminus. Synaptotagmins I and II probably serve as Ca2+ sensors in fast Ca2+-dependent exocytosis (10, 11). Synaptotagmin III may have a distinct function at the synapse, possibly in mediating the slow component of Ca2+-dependent release (12), while the other synaptotagmins are thought to function in related neuronal and nonneuronal membrane trafficking reactions (6). However, the total number of synaptotagmins and their localizations and functions are unknown. The current study was initiated to determine if there are additional synaptotagmins in mammals that could be grouped into subclasses with distinct properties.

Most synaptotagmins are Ca2+-binding proteins (1). In synaptotagmin I, the first C2-domain (C2A-domain) binds to phospholipids and syntaxin as a function of Ca2+ (6, 13). The second C2-domain mediates the Ca2+-dependent binding of synaptotagmin to itself, leading to Ca2+-dependent homomultimers (14, 15). A crystal structure of the C2A-domain of synaptotagmin I revealed that it represents a compact domain composed of eight beta -strands forming two beta -sheets (16). Three sequence loops emerge from the top, and four from the bottom of the domain. Ca2+ binding to the C2A-domain stabilizes it (17) but does not induce a major conformational change (18, 19). Detailed studies of Ca2+ binding to the C2A-domain by NMR spectroscopy demonstrated that it binds at least two Ca2+ ions at a site formed by two of the top three loops (19). Ca2+ is coordinated by five aspartate residues, three of which coordinate both Ca2+ ions. Thus the C2-domain contains an unusual Ca2+-binding site, designated the C2-motif, that is formed by aspartate residues on discontinuous sequence loops.

More than 50 C2-domain sequences are present in the data banks, suggesting that it is a widespread domain (20). The C2-motif is conserved in many of these C2-domains that are thus likely to bind Ca2+ similarly to the C2A-domain of synaptotagmin I. In agreement with the binding of at least two Ca2+ ions to the C2A-domain of synaptotagmin I, Ca2+ cooperatively activates phospholipid binding to native synaptotagmin I (12, 21) and to recombinant C2A-domain (13). Phospholipid binding is promiscuous and only requires negatively charged phospholipids (12). In addition to phospholipids, C2A-domains bind syntaxin as a function of Ca2+ but with distinct Ca2+ affinities that suggest different functions in membrane traffic (6).

Although most C2A-domains from synaptotagmins contain the C2-motif and bind phospholipids as a function of Ca2+, those of synaptotagmins IV and VIII do not (1, 6, 11). Synaptotagmin VIII contains many changes in the sequences of the Ca2+-binding loops, indicating that the C2-motif is not formed in this synaptotagmin. By contrast, the C2A-domain of synaptotagmin IV is very similar to that of the other synaptotagmins and contains all residues of the C2-motif except that one of the five Ca2+-coordinating aspartates is substituted for a serine. In this study, we now describe the identification of a novel synaptotagmin, synaptotagmin XI, that is highly homologous to synaptotagmin IV and also contains the aspartate to serine substitution in the C2-motif. Since the changed aspartate coordinates both Ca2+ ions in the C2-motif, the substitution is predicted to abolish Ca2+ binding, and indeed no Ca2+-dependent phospholipid binding was observed with standard liposomes for the C2A-domain of synaptotagmin IV (11).

In addition to participating in Ca2+-dependent interactions, C2-domains from synaptotagmins also exhibit Ca2+-independent activities. For example, the second C2-domain of all synaptotagmins tested binds AP2, a clathrin adaptor protein complex involved in endocytosis (6, 22). Since this and other Ca2+-independent interactions of C2-domains are not regulated, it is difficult to determine if they are physiologically relevant. Data showing that some C2-domains do not exhibit Ca2+-dependent interactions do not actually prove that these C2-domains are indeed Ca2+-independent (6, 11). Such a demonstration could be obtained, however, if reversal of an inactivating amino acid substitution restored Ca2+-dependent properties. Indeed, the notion that the C2A-domain of synaptotagmin IV is a Ca2+-independent domain was challenged by a report of Ca2+-dependent binding of the C2A-domain of synaptotagmin IV to phospholipids consisting of 100% PS1 (23). It is thus important to establish if synaptotagmins IV and XI are Ca2+-independent because this would support a general function for C2-domains in Ca2+-independent reactions. Therefore, a further goal of the current study was to determine if the C2A-domains of synaptotagmins IV and XI are selectively inactivated in evolution as Ca2+-binding modules by a single substitution, or if these C2-domains contain additional changes that differentiate them from other synaptotagmins. Our data reveal that synaptotagmins IV and XI contain a single point mutation that selectively abolishes Ca2+ binding but leaves other properties of the C2-domain intact.


EXPERIMENTAL PROCEDURES

Cloning of synaptotagmin XI and sequence analysis

Searches of GenBank with a consensus sequence from the C-terminal domain of synaptotagmins and double C2-domain proteins (1) uncovered a human EST sequence (accession number D38522[GenBank]) that represents a novel member of this family. PCR primers were designed based on the human sequence (sequences: GCGGAATTCCAGGTAATCCTTATGTCAAGGTGAA[C, T]GT and GGCCGTCGACTAGTA CTCGCTCAGACTGTGCCA[C, T]TT[C, T,A, G]GC; letters in brackets indicate redundant positions) and used for PCRs with total rat brain cDNA to isolate the corresponding rat sequence (24). A single product of the correct size (0.325 kb) was obtained, verified by sequencing, and used to screen a rat brain cDNA library by standard techniques (24). Positive clones were sequenced, with two clones containing the entire coding region as judged by comparison with the sequence of synaptotagmin IV and the presence of in-frame stop codons in the longest clone. The cDNA sequence has been deposited in GenBankTM (accession number AF000423).

Construction of Expression Vectors and Purification of Recombinant Proteins

The C2A-domains of synaptotagmins IV and XI were subcloned into pGEX-KG (25) with wild type or mutant sequence by PCR as described (6, 14), resulting in the following expression plasmids. pGEX-T1082/1083 and pGEX-T1082/1083S244D encode residues 151-282 of wild type and mutant synaptotagmin IV, respectively, with the mutant containing a substitution of serine 244 to aspartate; pGEX-T1454/1455 and pGEX-T1454/1455S247D encode residues 153-287 of wild type and mutant synaptotagmin XI, respectively, with the mutant containing a substitution of serine 247 to aspartate; both synaptotagmin XI constructs contain an additional C-terminal serine residue before the termination codon; and pGEX65-4 encodes residues 140-267 of synaptotagmin I. Recombinant GST-fusion proteins were purified on glutathione-agarose. Proteins were used for phospholipid binding measurements immobilized on glutathione-agarose without elution. For the CD studies, the C2A-domains were cleaved from the GST-fusion proteins on the column by thrombin (0.25 mg/ml of resin at room temperature for 3 h) and further purified by gel filtration on a Superdex-75 FPLC column.

Phospholipid Binding Measurements

Phospholipids (3.5 mg total, obtained from Avanti Poalr Lipids) were dissolved in chloroform, mixed in the indicated weight ratios with a trace amount of 3H-labeled PC (<0.01% total; Amersham), and dried under a stream of nitrogen. Dried lipids were resuspended in 20 ml of 50 mM HEPES-NaOH, pH 7.4, 0.1 M NaCl (buffer A) by vigorous shaking for 1 min. Suspensions were sonicated for 20 s in a Branson probe sonicator (model 450) at an intensity setting of 5 and centrifuged for 20 min at approximately 5,000 × g to remove aggregates. The standard binding assay contained 25 µg of recombinant protein with 1 µg of protein/µl wet glutathione beads. Beads were prewashed and resuspended in the respective incubation buffers (0.1 ml buffer A containing 1 mM EGTA, approx 9 µg of phospholipids with approx 0.025 µCi 3H-labeled PC, and either no additions or 1.12 or 2 mM either Mg2+ or Ca2+ as stated in the legend to Figs. 5 and 6). The mixture was incubated for 10 min at room temperature with vigorous shaking, briefly centrifuged, and washed eight times with 0.8 ml of the respective incubation buffer. Phospholipid binding was quantified by scintillation counting.


Fig. 5. Effect of Ca2+ on the temperature-dependent denaturation of the wild type and mutant C2A-domains from synaptotagmins IV and XI. Purified recombinant wild type C2A-domains from synaptotagmins IV and XI (A and C, respectively) and mutant C2A-domains from the same synaptotagmins (B and D, respectively) were incubated with 0.5 mM EGTA in the absence of Ca2+ (open circles) or in the presence of 5.5 mM Ca2+ (closed circles). Unfolding of the domains as a function of temperature was monitored by CD absorbance at conformation-dependent wavelength (217 nm). The mutants contain a single amino acid substitution exchanging the serine in the C2-motif for aspartate (see Figs. 3 and 4).
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Fig. 6. Ca2+-dependent phospholipid binding to wild type and mutant C2A-domains from synaptotagmins I, IV, and XI. GST alone and the C2A-domain of synaptotagmin I (GST-Syt I) were used as negative and positive controls, respectively (A). C2A-domain fusion proteins for wild type and mutant synaptotagmins IV and XI (B-E; GST-Syt IV and GST-Syt XI; superscript identifies mutation) were studied in standard liposome binding assays (13). Experiments in A-C were performed with liposomes composed of 29% PS, 71% PC; in D with 80% PS, 20% PC liposomes; and in E with 100% PS sonicated lipid suspensions. GST-fusion proteins were immobilized on glutathione beads and incubated with 3H-labeled liposomes in the presence of 1 mM EGTA with or without additions of divalent cations (2 mM Mg2+ or Ca2+ in A-C and 1.12 mM in D and E to reproduce the concentrations in Fukuda, et al. (23). Open bars, EGTA; cross-hatched bars, Mg2+; solid bars, Ca2+.
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Ca2+-dependent Thermal Denaturation Monitored by Circular Dichroism

Thermal denaturation data were obtained on an Aviv model 62DS spectropolarimeter using a 1-mm path-length cell. Approximately 8 µM C2A-domain in 40 mM Tris-HCl, pH 8.0, 0.1 M NaCl and 0.5 mM EGTA with or without 5.5 mM CaCl2 were used. Thermal denaturation was monitored by changes in CD absorption at 217 nm from 25 to 95 °C in 1 °C steps. The fraction of unfolded protein at each temperature was calculated as (Iobs - If)/(Iu - If), where Iobs is the observed signal intensity and Iu and If are the signal intensities of the unfolded and folded states, respectively. Iu and If were obtained by extrapolation of the linear regions of the unfolding curves.

Miscellaneous Procedures

Amounts of proteins used were standardized based on Coomassie Blue stained SDS-gels and UV absorption at 280 nm. RNA blotting experiments were performed at high stringency with blots loaded with poly(A)+-enriched RNA purchased from CLONTECH as described previously (24).


RESULTS

Cloning of Synaptotagmin XI

At the C terminus, synaptotagmins and other double C2-domain proteins contain a unique conserved domain after the second C2-domain (1). Searches of GenBankTM with a consensus sequence derived from the C-terminal domain identified a human EST sequence (accession number D38522[GenBank]) from a myeloblast cell line that was related to, but distinct from, all currently known synaptotagmins and other double C2-domain proteins. To determine if a homologue of this EST was present in rat brain, we synthesized PCR primers based on the human sequence and amplified a corresponding fragment from rat brain total cDNA. A single band of the correct size was obtained that was confirmed by sequencing to correspond to the rat homologue of the human EST sequence (data not shown). Using the PCR product as a probe, we isolated multiple independent cDNA clones from a rat brain cDNA library. Two of the cDNA clones were found to contain the complete coding region, and the sequence of the entire protein was assembled from the sequences of the cDNA clones (Fig. 1).


Fig. 1. Sequence of synaptotagmin XI and alignment with synaptotagmin IV. Sequences are from rat and shown in single letter amino acid code. Sequences are numbered on the right. The transmembrane region is shown in bold, and the two C2-domains are underlined. Their positions are also identified on the right. Residues that are identical in the two synaptotagmins are marked by an asterisk.
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The amino acid sequence translated from the cDNA sequence identifies the new double C2-domain protein as a synaptotagmin based on the criteria established for this protein family (1). It has no signal sequence, contains a single transmembrane region with multiple neighboring cysteines, has two C2-domains, and has a short C-terminal sequence homologous to the C-terminal domains of other double C2-domain proteins (Fig. 1). In continuation of the naming of other members of this protein family, we named this protein synaptotagmin XI.

Expression of Synaptotagmin XI

To learn which rat tissues express synaptotagmin XI, we performed an RNA blot analysis. High levels of synaptotagmin XI mRNA were observed in brain (Fig. 2). In addition, low levels of the mRNA could be detected in almost all tissues tested, suggesting that synaptotagmin XI, similar to synaptotagmins IV, VI, and VII (6), is not brain-specific but ubiquitously expressed in low abundance.


Fig. 2. RNA blot analysis of synaptotagmin XI expression. A blot containing poly(A)-enriched RNA from the indicated rat tissues (obtained from CLONTECH) was hybridized at high stringency with a synaptotagmin XI cDNA probe and exposed to film for 48 h. The arrow marks position of the synaptotagmin XI mRNA. kb, kilobases.
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Definition of a Subclass of Synaptotagmins

With the new synaptotagmin described here, synaptotagmins now constitute a family of 11 proteins (2-9). Although all synaptotagmins are composed of similar domains, their N-terminal regions (intravesicular sequence, transmembrane region, and linker between the transmembrane region and the C2A-domain) exhibit little similarity to each other except for pairwise similarities between synaptotagmins I and II and between III and VI (1, 6). By contrast, the C-terminal domains (the two C2-domains and the short C-terminal domain) are highly homologous between all synaptotagmins. Sequence analysis of synaptotagmin XI demonstrates that it is most closely related to synaptotagmin IV (53% overall identity; Fig. 1). Synaptotagmins IV and XI are homologous to each other in their N-terminal 73 amino acids which exhibit no sequence similarity to other synaptotagmins, defining them as a separate subgroup. In addition, the C2-domains of synaptotagmins IV and XI are more closely related to each other than to those of other synaptotagmins. The sequence differences between different synaptotagmins are not random variations since they are evolutionarily conserved (98% identity between rat and mouse synaptotagmin IV) (11). Similarly, the C-terminal 104 amino acids of human synaptotagmin XI encoded by EST D38522[GenBank] exhibits only a single amino acid change compared with the rat sequence.

The C2A-domain of synaptotagmin I binds two Ca2+ ions through five aspartate residues located in two loops (19). Three of the aspartates are bifunctional and ligate both Ca2+ ions. Alignment of different C2A-domains shows that most contain either aspartates or glutamates at the five positions that are involved in Ca2+ binding (Fig. 3). The C2A-domains of synaptotagmins IV and XI, however, are different. The general spacing is the same, but, in one position (corresponding to aspartate 230 in synaptotagmin I), a serine is substituted for the aspartate (Fig. 3). The aspartate to serine substitution appears not to be a sequencing or cloning artifact since it is present in both synaptotagmin IV and XI and since the serine is evolutionarily conserved in synaptotagmin IV (11).


Fig. 3. Alignment of the sequences of the C2-motif from the C2A-domains of synaptotagmins, doc2s, and rabphilin and from the C2-domain of protein kinase beta . Sequences are shown in single letter amino acid code and are identified on the left. SI to SXI, synaptotagmins I to XI; Rb, rabphilin; and PKCbeta , protein kinase Cbeta . Hyphens indicate gaps. The sequences from synaptotagmins XI and IV are shown on top in bold typeface. Aspartate and glutamate residues corresponding to the five aspartate residues in the C2A-domain of synaptotagmin I that mediate Ca2+ binding are shown on a shaded background. The position of the aspartate to serine substitution in synaptotagmins IV and XI is marked by an asterisk.
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Aspartate 230 is a critical residue in synaptotagmin I since it coordinates both Ca2+ ions and is at the center of the C2-motif (19). Modeling of the analogous Ca2+-binding site in synaptotagmins IV and XI suggests that in the presence of serine instead of aspartate, Ca2+ binding is unlikely (Fig. 4A). This model also suggests that Ca2+ binding could potentially be restored if the serine is mutated to aspartate (Fig. 4B). To test these hypotheses, we constructed expression vectors that direct the synthesis of GST-fusion proteins with the C2A-domains from synaptotagmins IV and XI either in the wild-type forms containing serine or in mutant forms containing aspartate instead of serine at the appropriate position.


Fig. 4. Model of the Ca2+-binding site in the C2-motif of the C2A-domain from synaptotagmin XI as wild type sequence containing serine (A) and as mutant with the serine to aspartate substitution (B). The diagram is based on the bipartite Ca2+-binding motif modeled for the C2A-domain of synaptotagmin I (19); residues are identified by numbers (Fig. 1). In the wild type form (A), serine 247 is predicted to be unable to act as a bidentate ligand for the two Ca2+ ions. In the serine to aspartate substitution mutant (B), aspartate 247 can contribute to bind the two Ca2+ ions as in the C2A-domain of synaptotagmin I. An analogous model is proposed for synaptotagmin IV, with the corresponding changes in residue numbers. Dashed lines illustrate the coordination of the two Ca2+ ions. The protein backbone linking aspartate residues in the same loop is represented by solid curves.
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Substitution of Serine to Aspartate in the C2-motif of the C2A-domain of synaptotagmins IV and XI Restores Ca2+ Binding

The GST-fusion proteins encoding the C2A-domains of synaptotagmins IV and XI either as wild type proteins or with serine to aspartate substitutions were isolated, the linkers between GST and the fused C2A-domain were cut with thrombin, and the C2A-domains were purified to homogeneity (data not shown). As a test of Ca2+ binding, we used temperature denaturation experiments since binding of a ligand to a protein usually leads to stabilization of the protein structure. Temperature denaturation can be monitored by CD measurements at a conformation-dependent wavelength. This provides a sensitive method to analyze Ca2+ binding to C2-domains as shown previously for the wild type and mutant C2A-domains from synaptotagmin I (18, 19).

Wild type C2A-domains from synaptotagmins IV and XI exhibited no change in denaturation temperature as a function of Ca2+ (Fig. 5, A and C), confirming that these C2-domains do not bind Ca2+ at concentrations of up to 5 mM although the data do not exclude the possibility of a binding site with even lower affinity. By contrast, C2A-domains carrying a single amino acid substitution that exchanged serine for aspartate at the position corresponding to aspartate 230 in synaptotagmin I experience Ca2+-dependent shifts in denaturation temperature (approximately 15 and 9 °C for synaptotagmins IV and XI, respectively; Fig. 5, B and D). Thus, the single amino acid substitution restores the ability to bind Ca2+ to these C2-domains.

Ca2+-dependent Phospholipid Binding Properties of Wild Type and Mutant C2A-domains from Synaptotagmins IV and XI

The C2A-domains of most synaptotagmins but not of synaptotagmin IV bind phospholipids as a function of Ca2+, whereas Mg2+ is ineffective (6, 11). This agrees well with the aspartate to serine substitution in the C2-motif of the C2A-domain of synaptotagmin IV (Fig. 4). The questions now arise if synaptotagmin XI, with a similar substitution, is also unable to bind phospholipids as a function of Ca2+ and if mutating the wild type serine back to aspartate would confer Ca2+-dependent phospholipid binding to these two C2A-domains.

Fusion proteins containing the wild type and mutant C2A-domains were immobilized on glutathione beads and used for phospholipid binding measurements. As shown in Fig. 6, A-C, no Ca2+-dependent binding of phospholipids consisting of 29% PS and 71% PC was observed to the wild type C2A-domains from synaptotagmins IV and XI. In contrast, the C2A-domain of synaptotagmin I exhibited robust Ca2+-specific phospholipid binding. No Ca2+-independent phospholipid binding was observed as the background level of phospholipids bound corresponds to that obtained with GST alone. The mutant C2A-domains, however, exhibited marked Ca2+-dependent phospholipid binding that was not observed with Mg2+ (Fig. 6). These data suggest that the C2A-domains of the synaptotagmin IV/XI subclass contain a selective, evolutionarily conserved single amino acid substitution that inactivates Ca2+ binding and Ca2+-dependent phospholipid binding.

Dependence of Ca2+-dependent Phospholipid Binding on Liposome Composition

Recently, it was reported that the C2A-domain of synaptotagmin IV can bind 100% PS in a Ca2+-dependent manner although it does not bind to liposomes composed of mixtures of PS and PC (23). Given the structure of the Ca2+-binding site in C2-domains (Fig. 4), this is a surprising finding since the C2A-domain is not expected to bind Ca2+ with the aspartate to serine substitution. However, PS itself binds Ca2+ at the concentrations used, and it is possible that clusters of negative charges by PS may also allow Ca2+ binding to the protein with unanticipated properties. Furthermore, stable bilayers are difficult to obtain with 100% PS, which tends to aggregate. Since the assay used by Fukuda et al. (23) utilized soluble GST-fusion proteins that were co-precipitated with lipids, the possibility arises that Ca2+-dependent PS aggregation could artifactually trap the C2A-domain.

To test if 100% PS induces a novel Ca2+-binding site in wild type synaptotagmin IV, we investigated the Ca2+-dependent interactions of the wild type and mutant C2A-domains from synaptotagmin IV with 100% PS in an assay that does not depend on lipid co-precipitation to avoid artifacts. No binding of 100% PS to the wild type C2A-domain was observed (Fig. 6, D and E). Even with the mutant C2A-domain, which exhibits robust Ca2+-dependent binding to liposomes composed of 29% PS, 71% PC, Ca2+ only had an insignificant effect on binding of 100% PS. Since, in the C2A-domain of synaptotagmin I, only negatively charged phospholipids bind as a function of Ca2+, the absence of Ca2+-dependent binding of 100% PS to the mutant C2A-domain of synaptotagmin IV is paradoxical. This result can best be explained by the assumption that a stable lipid bilayer may be required. To test this hypothesis, we made liposomes from 80% PS, 20% PC, which are more likely to contain stable bilayers. Now, Ca2+-dependent phospholipid binding to the mutant C2A-domain was observed although it was not as strong as that observed with our standard liposomes. The wild type C2A-domain, however, was still unable to bind. Together, these data provide further evidence for the conclusion that the wild type C2A-domain of synaptotagmin IV is not a Ca2+-binding domain and suggest that, even under conditions of PS enrichment, no Ca2+-dependent properties can be demonstrated.


DISCUSSION

Synaptotagmins form a large family of genes with putative functions in membrane traffic (1). We now report the structure and properties of the 11th member of this family. Since it seems likely that additional synaptotagmins remain to be discovered, synaptotagmins now form one of the largest protein families in membrane traffic second only to rab proteins.

The structure of synaptotagmin XI reveals that it defines a new subclass of synaptotagmins together with synaptotagmin IV. Synaptotagmins IV and XI are highly expressed in brain, and at lower levels in other tissues (Fig. 2; see Ref. 6). This indicates a function that is concentrated in brain but also operative in nonneural tissues. Synaptotagmins I and II probably serve as Ca2+ sensors in neurotransmitter release (10, 11), suggesting that the other synaptotagmins also represent membrane trafficking proteins at the plasma membrane, possibly in regulating different forms of exocytosis. In addition, synaptotagmins may function in endocytosis since all synaptotagmins tested are high affinity binding proteins for the clathrin adaptor protein complex, AP2 (6, 11, 22).

The subgroup of synaptotagmins composed of synaptotagmins IV and XI is characterized by a homologous N-terminal region and by identical deviations from the C2-domain consensus sequence shared by most synaptotagmins (Figs. 1 and 3). The most remarkable change in the C2-domains of synaptotagmins IV and XI is the substitution of one of the aspartates of the Ca2+-binding site for a serine. This raises the question if a C2A-domain with this substitution is capable of Ca2+ binding. We show by two assays, the Ca2+-dependent shift in the heat denaturation curve and Ca2+-dependent phospholipid binding, that Ca2+ has no effect on the C2A-domains from both synaptotagmins at concentrations up to 5 mM. A point mutation that reverses the evolutionary serine to aspartate substitution, however, restores both Ca2+-dependent properties to both synaptotagmins. This result indicates that this amino acid change is the only change in the C2A-domain that made the C2A-domains from synaptotagmins IV and XI Ca2+-independent. Thus, this subgroup of synaptotagmins is characterized by an evolutionarily conserved substitution in the C2A-domain that abolishes Ca2+ binding.

The most amazing aspect of our findings is that evolution seems to have selected for the inactivation of Ca2+-binding in two different synaptotagmins by a single amino acid substitution while leaving the remaining structure intact. Based on their overall sequence similarity, it seems likely that synaptotagmins IV and XI share a common ancestor derived from the original "ur-synaptotagmin". Further evolution then created the diversification into synaptotagmins IV and XI with many amino acid changes, including several in their C2A-domains. Nevertheless, these changes did not abolish the ability of these synaptotagmins to bind Ca2+ and to exhibit Ca2+-dependent phospholipid binding if only the serine in the Ca2+-binding site was rechanged into aspartate.

The structure of the C2-domain consists of a stable core composed of two beta -sheets with three loops emerging on top and four on the bottom (16). Ca2+ binding occurs only to the top and induces no electrostatic or conformational change in the bottom (18, 19). It is possible that C2-domains serve as janus-faced interaction domains, a Ca2+-dependent top and a Ca2+-independent bottom (1). This would imply that in the synaptotagmin IV/XI subgroup, evolutionary pressure led to a selective retention of only the Ca2+-independent properties. Unfortunately no Ca2+-independent activities of a C2A-domain have been identified yet. Once these have been discovered, however, it will be important to test them in different synaptotagmins to determine if they are selectively retained in the Ca2+-independent forms.

In a very interesting study, synaptotagmin IV was identified as an immediate early gene (26). This suggests that a switch from Ca2+-dependent to Ca2+-independent synaptotagmins may occur during strong stimulation of neurons. Such a switch could be particularly useful during pathological hyperexcitation that is accompanied by unimpeded Ca2+ influx. A switch to a Ca2+-unresponsive synaptotagmin under those conditions would eliminate a Ca2+ target and maybe inhibit excessive neurotransmitter release. Future studies will have to test this hypothesis and also investigate if synaptotagmin XI is also an immediate early gene.


FOOTNOTES

*   This study was supported in part by National Institutes of Health Grant R29-NS33731 (to J. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   Recipient of a Fellowship from the Max-Planck-Society.
**   To whom correspondence should be addressed. Tel.: 214-648-5022; Fax: 214-648-6426.
1   The abbreviations used are: PS, phosphatidylserine; EST, expressed sequence tag; GST, glutathione S-transferase; PC, phosphatidylcholine; PCR, polymerase chain reaction.

ACKNOWLEDGEMENTS

We thank A. Roth, E. Borowicz, and I. Leznicki, for excellent technical assistance, and Drs. M. S. Brown and J. L. Goldstein, for advice.


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