Calcium-dependent Interaction of the Cytoplasmic Region of Synaptotagmin with Membranes AUTONOMOUS FUNCTION OF A SINGLE CZ-HOMOLOGOUS DOMAIN*

The synaptic vesicle protein synaptotagmin has been implicated in the docking and subsequent calcium-regu-lated exocytosis of synaptic vesicles. We demonstrate that synaptotagmin is a major constituent of synaptic vesicle membranes, comprising 74% of the total vesicle protein. A proteolytic fragment of synaptotagmin, containing two repeats homologous to the (&-domain of protein kinase C, bound to a variety of natural membranes in a calcium-dependent manner (ECso - 30 p~ calcium). Binding was insensitive to proteolysis of the acceptor membranes suggesting an interaction with the lipid con-stituents. This interaction was confirmed using a recom- binant fusion protein, containing both C2-like domains of synaptotagmin, that bound to artificial liposomes in a calcium-dependent manner. Phospholipid binding properties were preserved in a 114-amino acid domain corresponding to the first Cz-like repeat of the protein and represents the shortest functional cassette yet reported. Furthermore, deletion of a highly conserved 9-amino acid motif, within this region, was sufficient to abolish the calcium-dependent phospholipid binding properties of this domain. This mutation may provide a means to selectively disrupt individual C2-domains in order to as-sess their relative contributions to function.

the protein has been addressed in a number of recent studies. For instance, calcium-dependent secretion from large densecore vesicles occurs in PC12 cell lines lacking synaptotagmin (Shoji-Kasai et al., 1992). Mutations in the synaptotagmin genes of Caenorhabditis elegans (Nonet et al., 1993) and Drosophila (DiAntonio et al., 1993;Littleton et al., 1993) result in severely impaired nervous system function, consistent with defective synaptic transmission. These studies indicate that synaptotagmin is not an absolute requirement for exocytosis to occur but suggest that the protein plays a crucial modulatory role in regulated secretion. This role has been examined by Littleton et al. (1993) who reported that Drosophila larva, with partial lack-of-function synaptotagmin mutations, exhibited an increase in miniature excitatory potentials and a decrease in calcium-induced neurotransmitter release.
The most striking structural feature of synaptotagmin is the presence of two repeats, in the cytoplasmic domain, which are homologous to the C2-regulatory domain of protein kinase C (Perin et al. (19901, see also Fig. 2). C2-domains are conserved regions shared by calcium-dependent but not calcium-independent isoforms of protein kinase C, suggesting that these regions confer calcium dependence (reviewed by Nishizuka (1988)). Homologous domains have subsequently been identified in other proteins which interact with lipids including cytosolic phospholipase A2 (Clark et al., 1991), phospholipase C-y,) (Stahl et al., 19881, GTPase-activating protein (Vogel et al., 1988), and rabphilin (Shirataki et at., 1993). A consensus sequence within this conserved domain has been reported by Clarket al. (1991) and is given in Fig. 2. Recent studies indicate that the Cz-like domains of synaptotagmin (referred to as Czdomains) may be involved in the regulation of calcium-dependent exocytosis. For instance, microinjection of a recombinant fragment containing the first CP-domain of synaptotagmin inhibited calcium-dependent exocytosis of large dense core vesicles in PC12 cells (Elferink et al., 1993). Peptides corresponding to sequences within the Cz-domains also inhibited neurotransmitter release when injected into squid giant presynaptic nerve terminals (Bommert et ai., 1993).
While such findings suggest that the C2-domains are important for the function of synaptotagmin, surprisingly little is known regarding their biochemical properties. For instance, a deletion encompassing most of the C2-domain of protein kinase Ca failed to abolish calcium and phospholipid dependent activity (Kaibuchi et al., 1989). In addition, limited proteolytic cleavage of synaptotagmin (see Fig. 2) appeared to abolish calciumdependent lipid interactions (Brose et al., 1992) despite the fact that the C,-domains remained intact. These findings question whether Cz-domains are solely responsible for the calcium/ phospholipid-binding properties of their parent molecules.
In the present study, we have examined the interaction of synaptotagmin with membranes, with particular emphasis on the function of its C2-domains. Reconstitution of the cytosolic 5735 domain of the protein with native membrane preparations revealed a calcium-dependent interaction. Using recombinant fragments of synaptotagmin and artificial liposomes, we observed that the cytoplasmic domain of synaptotagmin selectively binds to negatively charged phospholipids in a calciumdependent manner. In addition, these properties were preserved in a recombinant fusion protein which contained only the first C2 repeat of synaptotagmin. Furthermore, we identify a 9-amino acid motif crucial for the binding properties of this domain.

EXPERIMENTAL PROCEDURES
Purification of Proteins-Synaptotagmin was purified by affinity chromatography using the anti-synaptotagmin monoclonal antibody 41.1 as described by Brose et al. (1992). Protein concentrations for purified synaptotagmin and the membrane preparations described below were determined using the Pierce BCA (bicinchoninic acid) reagents.
Reconstitution of the Cytoplasmic Domain of Synaptotagmin with Membranes-A tryptic fragment comprising most of the cytoplasmic domain of synaptotagmin was prepared by limited proteolysis of highly purified SV membranes. SVs were purified according to Huttner et al. (1983) and digested with trypsin at a trypsin:vesicle membrane protein ratio of 1:400 (w/w) for 15 min a t room temperature. Digestions were terminated by the addition of a 50-fold excess (w/w) of soybean trypsin inhibitor, 2 m~ phenylmethylsulfonyl fluoride, 10 p~ aprotinin, 10 pg/ml pepstatin A, and 10 1.1~ leupeptin. To examine the interaction of the fragment with purified SV membranes, 25 pg of the trypsin-treated SVs were aliquoted in a final volume of 100 p1 of 10 mM HEPES, pH 7.4, 150 mM NaCI, 2 mM MgCI,, and 1 m~ calcium or 2 m~ EGTA. In experiments in which the calcium concentrations were titrated, the free calcium concentrations were buffered with EGTA and calculated using software developed by Fohr et al. (1994). Samples were incubated for 30 min on ice with occasional mixing and pelleted by centrifugation at 50,000 rpm for 35 min using a Beckman TLA 100.3 rotor. For reconstitution with other membranes, 1 mM EGTA was added to the tryptic digest and the vesicle membranes were removed by centrifugation as described above. Supernatant containing the soluble tryptic fragment was mixed with other membrane preparations (described below) and assayed for binding, again, by co-sedimentation. In these experiments, cytosolic fragment derived from 25-pg SV membranes was reconstituted with 50 pg of LP1 or COS-7 acceptor membranes or 25-pg SV membranes. Following sedimentation of the 39-kDa fragment-membrane complex, supernatants were removed and the pellets solubilized in an equal volume of 1% SDS. An equal volume of 3 times SDS sample buffer (180 m~ Tris, pH 6.8, 6% SDS, 30% sucrose, and 10% P-mercaptoethanol) was added to each sample followed by boiling for 3 min. Samples were stored at -20 "C or immediately subjected to SDS-PAGE and immunoblot analysis.
A synaptosomal plasma membrane-enriched preparation (LP1) was prepared essentially as described by Huttner et al. (1983). Briefly, adult rat brain synaptosomes were lysed by hypotonic shock in 15 volumes of distilled water and homogenized 6 times a t 2,000 rpm in a Teflon homogenizer. The heavy membranes (LP1) were collected by centrifugation at 12,000 x g for 10 min. COS-7 membranes were prepared by suspending cells in 5 m~ HEPES and homogenizing with 80 strokes in a 5-ml Teflon homogenizer. Nuclei and unbroken cells were removed by centrifugation at 500 x g for 5 min, membranes were then collected by centrifugation at 12,000 x g for 10 min. Synaptosomal and COS-7 membranes were adjusted to 10 mg/ml and stored a t -70 "C. An aliquot of each of these membrane preparations, as well as purified SV membranes, was digested with 10% (w/w) trypsin and 10% (w/w) chymotrypsin for 4 h at 37 "C. Proteases were then inhibited by boiling for 3 min followed by the addition of soybean trypsin inhibitor (50 fold, W/W, excess), 1 m~ tosylphenylalanyl chloromethyl ketone, 10 m~ phenylmethylsulfonyl fluoride, 1 pg/pl aprotinin, and 33 PM leupeptin. Membranes were stored at -70 "C.
Immunoblot Analysis-All SDS-PAGE was carried out using the Bio-Rad Mini Protean Gel I1 apparatus. Mouse monoclonal antibody 41.1, raised against a recombinant cytoplasmic domain of synaptotagmin has been described earlier (Brose et al., 1992). A new set of monoclonal antibodies was generated by standard procedures (Kohler and Milstein, 1975;Jahn et al., 1985). Using a 12-amino acid synthetic peptide derived from the amino terminus of rat synaptotagmin I (CAWSASH-(C1 604.14).
Blots probed with lZsI-protein A were blocked overnight in blotto. Primary antibodies were added as above and incubated for 1 h a t room temperature. Blots were washed 5 times, incubated for 1 h with rabbit anti-mouse IgG antibodies (Cappel) diluted 1:1000, washed 5 times, and incubated with 0.1 pCi/ml lz5I-protein A (DuPont-New England Nuclear) for 1 h, washed 5 times, dried, and autoradiographed.
Preparation of Recombinant Fusion Proteins-cDNA encoding rat synaptotagmin I was kindly provided by T. C. Sudhof (Dallas, Tx; Perin et al., 1990). All polymerase chain reactions (PCR) were carried out using Pfu polymerase (Stratagene Corp.). The C2ABs-GST and C2A-GST fusion constructs were prepared by generating the nucleotide sequences 291-1263 (amino acid residues 97-421) and 402-744 (amino acid residues 135-248), respectively, using PCR primers flanked by EcoRI restriction sites. The PCR products were fused in frame to the COOH terminus of glutathione S-transferase by ligating into the EcoRI site of pGEX-1 (Amrad Corp., Ltd.). The region encoding amino acid residues SDPYVKVFL (base pairs 529-555) was deleted from the C2A domain (designated C 2 M ) using the overlapping primer method of Higuchi (1990). Briefly, PCR was carried out using an antisense oligonucleotide, complimentary to 18 base pairs flanking each side of the region to be deleted, and the 5' primer used to prepare C2A. In a separate PCR, a sense oligonucleotide complimentary to the same 18 base pairs flanking each side of the region to be deleted and the 3' primer used to prepare C2A were used. These PCR products were gel purified and annealed. The full-length deletion mutant was then generated by carrying out PCR between the annealed region and the 5' and into the EcoRI site of pGEX-1. JM109 cells expressing GST, C2ABs-3' end primers used to prepare C2A. Again, this product was subcloned GST, C2A-GST, or C2AA-GST were grown to an A,,, of 0.4, induced by the addition of 0.4 m M isopropyl-1-thio-P-o-galactopyranoside, and harvested after 4 h. Cells were pelleted, resuspended in 10 ml of 20 m M Tris, pH 7.2,150 m M NaCl (TS buffer), sonicated three times for 30 s, and the suspension centrifuged a t 12,000 rpm for 20 min in an SS34 rotor. The supernatant was incubated with 0.5 ml of glutathione-Sepharose 4B (Pharmacia) for 15 min at 4 "C with mixing, poured into a column, and washed with 20 ml in TS and stored as a 50% slurry in TS buffer a t 4 "C. Aliquots were subjected to SDS-PAGE, visualized by Coomassie Blue staining, and the concentration of the fusion proteins determined by comparison with GST standards.
Lipids were mixed in the desired proportions, dried under a stream of nitrogen, and placed under vacuum for 60 min. The dried lipids were resuspended in TS buffer by vigorous vortexing using glass beads and liposomes were formed by sonication in a Branson Cup Sonicator, intensity setting 7, a t 50% duty cycle, for 8 min. Large aggregates and multilamellar liposomes were removed by centrifugation at 15,000 X g for 10 min. Liposome binding assays were carried out in USB Compact Reaction Columns with 0.35-pm filters with GST and the GST fusion proteins immobilized on glutathione-Sepharose 4B. Three hundred pg of phospholipids containing 1.44 x los dpm of c3H1PC were incubated with 0.8 pmol of C~ABS-GST, GST, C2A-GST, or C2AA-GST in TS buffer containing 2 mM MgCl, and 1 m M calcium or 2 m~ EGTA at 4 "C for 30 min with mixing. Samples were washed six times with 500 pl of 20 m~ Tris, pH 7.2, 200 m M NaCI, and 1 mM calcium or 2 m~ EGTA. Radioactivity was measured by liquid scintillation counting. All assays were carried out in triplicate. Calcium-independent phospholipid binding to immobilized recombinant synaptotagmin was dependent on the composition of the liposomes. For instance, C2ABs-GST bound a total of 7.1 x
dard curve, the abundance of synaptotagmin in the SV membrane lanes alone immobilized to glutathione-Sepharose bound 1.6-2.5 x IO3 dpm of labeled liposomes regardless of their composition or the presence of EGTA or calcium. The high degree of calcium independent binding of liposomes comprised of PE and PIPC is likely due to nonspecific ionic and hydrophobic interactions with the immobilized synaptotagmin fusion protein. To eliminate these variables we measured the calciumdependent component of the binding directly by elution with EGTA. Assays were carried out as described above. Following the washes, phospholipids which bound in a calcium-dependent manner were eluted by incubating the samples for 15 min in 200 pl of 20 m M Tris, pH 7.2.50 m M NaCI, and 10 mM EGTA a t 4 "C. The eluate was collected by centrifugation. Radioactivity in the entire eluate was measured by liquid scintillation counting. These data are plotted in Fig. 6.
Densitometry-Densitometry was carried out using a Visage 2000 scanner (Bio Image Products, MilligenEIioresearch Division of Millipore).

Synaptotagmin Is a Major Constituent of Synaptic Vesicle
Membranes--In the first set of experiments, we measured the amount of synaptotagmin in a highly purified preparation of SVs in order to determine its relative abundance with respect to other SV proteins. For this purpose, synaptotagmin, affinity purified as described (Brose et al., 19921, was used to generate a standard curve in a quantitative immunoblotting procedure (Fig. 1). From these data, synaptotagmin comprises 7-8% (w/w) of total vesicular protein. This is comparable to the abundance of synaptophysin (Jahn et al., 1987) and synapsin (Goelz et al., 1981), resulting in a stoichiometric ratio of synaptotagmin: synaptophysin of 0.74:l.

Reconstitution of Synaptotagmin JMembrane
Interactions-To study synaptotagmidmembrane interactions, we have prepared a tryptic fragment as well as recombinant proteins encompassing cytoplasmic domains of the molecule. It has been reported earlier that synaptotagmin contains a hypersensitive proteolytic cleavage site between residues 111 and 112 (Perin et al., 1991;Tugal et al., 1991) . 2. Schematic alignment of the synaptotagmin constructs and proteolytic fragments used in this study. A schematic representation of synaptotagmin is shown at the top: the transmembrane region ( 7 " ) and the two C, repeats (boxed) are indicated. The NHz terminus faces the lumen of the vesicle. Below, the fragments resulting from limited tryptic proteolysis are depicted. Cleavage occurs between residues 111 and 112 (Tugal et al., 1991;Perin et al., 1991). C2AJ3s and C2A represent the regions of synaptotagmin fused to GST and used in the epitope mapping and phospholipid binding studies. C2ABs corresponds to residues 97-421 and C2A, the first C,-domain of synaptotagmin, corresponds to residues 135-248 (Perin et al., 1990). Shown below is the Cz-domain consensus sequence for calcium-dependent lipid-binding proteins (Clark et al., et al., 1990). Bold letters indicate residues which are conserved relative to the consensus sequence. A, denotes the region deleted from C2Aresulting 1991). For comparison, the corresponding sequences from PKCa (Coussens et al., 1986)   lyzed by immunoblotting using mouse monoclonal antibody 41.1 (directed against the cytoplasmic domain) and a newly generated monoclonal antibody directed against the NH2-terminal 12 amino acids of synaptotagmin I (clone 604.4). As shown in Fig. 3  To further characterize the epitope recognized by the antibody 41.1, we prepared a set of synaptotagmin-glutathione Stransferase (GST) fusion proteins. The structure of these fusion proteins is summarized in Fig. 2. The construct C2ABs-GST encodes residues 97-421 and thus contains both C2-domains and the COOH terminus of synaptotagmin. C2A-GST contains the first Cp-domain (residues 135-248) as defined previously (Perin et al., 1990). C2AA-GST is identical to C2A-GST except that we have deleted a highly conserved motif (SDPYVKVFL, residues 177-185). As shown in Fig. 3B, 41.1 recognized C2ABs-GST and C2A-GST but did not react with C2AA-GST. These data demonstrate that residues 135-248 (C2A) contain the 41.1 epitope and that the sequence 177-185 contains residues critical for 41.1 recognition of either the primary or a higher order structure of synaptotagmin.
In the next series of experiments, we investigated the interactions of the 39-kDa cytoplasmic fragment of synaptotagmin with native membranes in a co-sedimentation assay. For comparison, we utilized SVs as well as synaptosomal and fibroblast membranes as the acceptor membranes. In the absence of calcium, only a small degree of binding (approximately 20%) to each of the target membranes was observed (Fig. 4, left). In the presence of calcium, the synaptotagmin fragment was quantitatively associated with the particulate fraction. This interaction appears to be specific for calcium with respect to magnesium since magnesium was included in all our buffers and failed to mimic the effects of calcium. To determine whether this binding reflected an interaction with proteins or lipids in the acceptor membrane preparations, these membranes were -. I . subjected to extensive proteolytic digestion followed by boiling prior to the co-sedimentation assay. As in the untreated membranes, calcium resulted in quantitative binding of the synaptotagmin fragment. These findings suggest that the fragment bound to the phospholipid components of the membrane. The residual membrane binding observed in the absence of calcium was abolished by protease treatment and boiling indicating that this component was mediated by proteidprotein interactions.
Since the calcium dependence for the association of synaptotagmin with liposomes is dependent on the phospholipid composition (Brose et al., 1992), the calcium concentration required for binding to native membranes has not been established. We therefore used SV membranes as the tryptic fragment acceptor and examined the calcium dependence for binding. We estimate that the effective concentration for 50% binding was approximately 30 p~ free calcium (Fig. 5A). Chelation of calcium prior to sedimentation completely reversed binding of the fragment to membranes (Fig. 5B), confirming our previous report (Brose et al., 1992). Addition of calcium in the absence of membranes did not result in sedimentation of the fragment demonstrating that sedimentation in the presence of membranes is not due to nonspecific aggregation of the fragment (Fig. 5C).
Calcium-dependent Phospholipid Binding to Recombinant Fragments of Synaptotagmin-The results described above suggest that a soluble cytoplasmic fragment of synaptotagmin, containing both C2-domains, interacts with membrane phospholipids in a calcium-dependent manner. To further analyze this interaction, we utilized the synaptotagmin-GST fusion proteins described above. To measure binding, all fusion proteins were immobilized on glutathione-Sepharose and mixed with radiolabeled artificial liposomes of defined compositions. Lipids which bound in a calcium-dependent manner were eluted by the addition of EGTA and quantified by liquid scintillation counting.
C~ABS-GST, the recombinant fusion protein which includes both C2-domains, bound to liposomes comprised of phosphatidylcholine/phosphatidylserine and phosphatidylcholine/phosphatidylinositol in a calcium-dependent manner. Calcium-dependent binding was not observed with liposomes composed of phosphatidylcholine alone or phosphatidylcholine/ phosphatidylethanolamine or when GST alone was immobilized to the beads (Fig. 6 A ) .
We further investigated the structural requirements for calcium-dependent phospholipid binding by utilizing the fusion protein containing only the first C2-domain of synaptotagmin. As shown in Fig. 6B, this 114-residue domain conferred calcium-dependent PS binding to GST, and thus represents the shortest defined functional calcium-dependent phospholipid binding motif. Within this domain, deletion of the highly conserved amino acid sequence (SDPYVKVFL) (see Fig. 2) abolished calcium-dependent phospholipid binding (Fig. 6R ). These findings suggest that the calcium-dependent phospholipidbinding domain lies in the NH2-terminal region of C2-domains and that the SDPYVKVFL sequence is essential for these properties.

DISCUSSION
In the present study, we have demonstrated that the cytoplasmic domain of synaptotagmin interacts with phospholipid membranes in a calcium-dependent manner. Recombinant protein containing most of the cytoplasmic domain also bound to phospholipids in a calcium-dependent manner indicating that this property is encoded in the primary sequence of the protein and is not dependent on post-translational modifications. This --f 3 9 k~a interaction was preserved in a recombinant GST fusion protein fragment which only contained the first C2-domain of synaptotagmin. This 114-residue region represents the shortest functional calcium-dependent phospholipid-binding domain reported to date. synaptotagmin/membrane interactions. The 3 9 -k~~ cytoso~ic do-Interestingly, the deletion of a highly Co~served stretch of 9 main of synaptotagmin was prepared by limited proteolysis of SV mem-amino acids within this region (consensus sequence S D P Wbranes and reconstituted with undigested SV, synaptosomal (LPI ) and -L), abolished calcium-dependent phospholipid binding activity.  (1991)) failed to abolish the calcium and phospho~ipid toradiography. Closed arrows indicate the endogenous intact synaptotarnin in the sv and Lpl membranes which is not expressed in cos-7 stimulated activity of the enzyme (Kaibuchi et al., 1989). Incells. The majority (80%) of the reconstituted 39-kDa fragment of syn-terestingly, the S D P w --L motif was the only Portion of the aototamin is soluble in the absence ofcalcium and becomes associated consensus seauence meserved in this deletion mutant (Kaibu- Calcium was added to yield the free calcium concentrations indicated. Membranes were sedimented and equal fractions of the supernatants (sn 1 and pellets ( p ) were subjected to SDS-PAGE and immunoblotted using the monoclonal anti-synaptotagmin antibody 41.1. Bands were visualized with '2511-protein A and autoradiography. The ECso for the calcium-dependent association of the 39-kDa fragment of synaptotagmin with membranes was approximately 30 p~ calcium. B, 15 min prior to sedimentation of the SV membranes, 5 m M EGTA was added to an aliquot of the SV digest which had been incubated with 1 mM free calcium. The addition of EGTA completely reversed the association of the 39-kDa fragment of synaptotagmin with membranes. C, to ensure that the calcium-dependent binding of the 39-kDa fragment of synaptotagmin to the particulate fraction was due to its association with membranes, supernatant containing the fragment was adjusted to 1 m M free calcium and centrifuged with the other samples. In the absence of membranes, the fragment did not sediment.

A B
FIG. 6. Calcium-dependent binding of SH-labeled phospholipid vesicles to recombinant domains of synaptotagmin. A, C~ABS-GST, a recombinant fusion protein containing the cytoplasmic domain of synaptotagmin (see Fig. 2) and GST alone, were immobilized on glutathione-Sepharose and assayed for calcium-dependent phospholipid binding as described under "Experimental Procedures." Samples were incubated in either 2 m~ EGTA (cross-hatched columns) or 1 mM calcium (open columns) in 20 mM Tris, pH 7.4, 150 mM NaC1, and 2 m~ MgClz with [3HlPC-labeled liposomes of the indicated composition. After washing, liposomes which bound in a calcium-dependent manner were eluted with 10 mM EGTA and quantified by liquid scintillation counting. In addition to the EGTA eluted phospholipid binding data plotted in the figure, the total amount of calcium-dependent and independent phospholipid binding was also determined and these data are given under "Experimental Procedures.'' Error burs represent standard deviations from triplicate measurements. C2ABs-GST bound, in a calcium-dependent manner, to liposomes containing PS and PI but not PE or PC. Calcium-dependent phospholipid binding was not observed in the GST control. B, phospholipid binding to a single C,-domain of synaptotagmin. C2A-GST was assayed for calcium-dependent binding to liposomes composed of PSPC as described in A. Deletion of a highly conserved 9-amino acid segment (see Fig. 2) abolished calcium-dependent PSPC binding. may provide an additional tool to perturb the function of synaptotagmin in microinjection experiments as described by Elferink et al. (1993).
The data presented here contradict our previous observations which suggested that proteolytic release of the cytoplasmic domain of synaptotagmin abolished binding activity (Brose et al., 1992). This discrepancy may be due to limitations in the assay system used to monitor binding in our previous study (fluorescence resonance energy transfer as compared to co-sedimentation used here). We assume that tryptic cleavage of synaptotagmin may have resulted in further degradation of the functional 39-kDa fragment such that the amount of energy donor fell below the limits of detection of the energy transfer assay system.
Our data show that synaptotagmin binds efficiently to membranes of neuronal and non-neuronal origin in a calcium-dependent manner, regardless of whether these membranes were pretreated with proteases. Two conclusions can be drawn from these findings: first, synaptotagmin binds to native membranes as well as to phospholipid vesicles and second, the binding is due to an interaction with membrane lipids with and does not require protein components. The calcium requirements for the membrane association was high approximately 30 VM) relative to other calcium-binding proteins. This is in agreement with the findings of Brose et al. (1992) using the intact protein and artificial liposomes which contained 25% acidic phospholipids. Such a low affinity for calcium was predicted for the exocytotic calcium receptor based on electrophysiological studies (Smith and Augustine, 1988;Llinas et al., 1992).
The data presented in this study clarify the structural requirements for the calcium-dependent phospholipid binding properties of synaptotagmin. To date, synaptotagmin is the only calcium-binding membrane protein which has been definitively demonstrated to be present in SVs. Its abundance ( 7 4 % of total vesicle protein) is consistent with a critical regulatory function in secretion as suggested by previous studies (Bommert et al., 1993;DiAntonio et al., 1993;Elferink et al., 1993;Littleton et al., 1993;Nonet et al., 1993). The 9-residue deletion which abolishes calcium-dependent phospholipid binding provides a means to selectively disrupt individual C2-domains. Such an approach may be utilized to determine the physiologi-cal function of these domains in, for instance, rescue experiments of synaptotagmin-deficient organisms.