ACCELERATED PUBLICATION
Mechanism of Calcium-independent Synaptotagmin Binding to Target
SNAREs*
Colin
Rickman and
Bazbek
Davletov
From the Medical Research Council Laboratory of Molecular
Biology, Hills Road, Cambridge CB2 2QH, United Kingdom
Received for publication, December 13, 2002
 |
ABSTRACT |
Synaptic vesicle exocytosis requires three SNARE
(soluble N-ethylmaleimide-sensitive-factor attachment
protein receptor) proteins: syntaxin and SNAP-25 on the plasma
membrane (t-SNAREs) and synaptobrevin/VAMP on the synaptic vesicles
(v-SNARE). Vesicular synaptotagmin 1 is essential for fast synchronous
SNARE-mediated exocytosis and interacts with the SNAREs in
brain material. To uncover the step at which synaptotagmin becomes
linked to the three SNAREs, we purified all four proteins from brain
membranes and analyzed their interactions. Our study reveals that, in
the absence of calcium, native synaptotagmin 1 binds the t-SNARE
heterodimer, formed from syntaxin and SNAP-25. This interaction is both
stoichiometric and of high affinity. Synaptotagmin contains two
divergent but conserved C2 domains that can act independently in
calcium-triggered phospholipid binding. We now show that both C2
domains are strictly required for the calcium-independent interaction
with the t-SNARE heterodimer, indicating that the double C2 domain
structure of synaptotagmin may have evolved to acquire a function
beyond calcium/phospholipid binding.
 |
INTRODUCTION |
Syntaxin, SNAP-25,1 and
synaptobrevin, also known as VAMP, are members of a large family of
SNARE proteins that likely execute fusion in all intracellular
compartments (1-3). Whereas most intracellular membrane fusion
reactions are constitutive, neurotransmission relies on the coupling of
neurotransmitter release to calcium influx into the nerve terminal (4).
On calcium entry, a proportion of synaptic vesicles fuse with the
presynaptic membrane and release neurotransmitter with a delay of less
than 1 ms (5). The synchronization of neuronal exocytosis to calcium
entry has been attributed to synaptotagmin, a major synaptic vesicle
protein (6, 7). Synaptotagmin contains two calcium-binding C2 domains
and is proposed to be a calcium sensor in neuronal exocytosis (8,
9).
Despite identification of the major players involved in
neurotransmitter release, the molecular mechanisms responsible for the tight coupling between calcium influx and synaptic vesicle exocytosis are still under debate (10, 11). Previous biochemical studies implicated calcium-dependent binding of
synaptotagmin to either syntaxin or SNAP-25 in vesicular exocytosis
(12-14). However, the short delay between calcium influx and the
fusion of synaptic vesicles suggests that synaptotagmin is linked to the fusion machinery prior to calcium influx into the nerve terminal. Indeed, synaptotagmin, in the absence of calcium, co-purifies with the
SNARE complexes from brain material as demonstrated using anti-syntaxin
immunoprecipitation and
-SNAP affinity chromatography (15, 16).
The calcium-independent molecular link between the calcium
sensor and the SNARE fusion machinery is not well understood, and the
previous studies using recombinant protein fragments yielded conflicting results (reviewed in Refs. 10 and 11). Therefore, we
investigated this link using, for the first time, highly purified brain
synaptotagmin and SNARE proteins as a starting point. We now
demonstrate that brain-purified t-SNAREs, syntaxin and SNAP-25, form a
stable heterodimer that can bind native synaptotagmin in the absence of
calcium. Since this interaction can be reproduced using the recombinant
cytoplasmic domain of synaptotagmin 1, we were able to address the
requirement for C2 domains in this binding. Our results show that the
calcium-independent binding of synaptotagmin to the t-SNARE heterodimer
requires the double C2 domain structure.
 |
EXPERIMENTAL PROCEDURES |
Isolation of SNARE Proteins and Synaptotagmin--
All
procedures were carried out at 4 °C. 1 mg of anti-SNAP-25 SMI 81 monoclonal antibody (Sternberger Monoclonals) was covalently coupled to
1 ml of CNBr-activated Sepharose-4B (Amersham Biosciences) according to the manufacturer's instructions. 3.5 g of bovine cerebral cortex was homogenized in 50 ml of phosphate-buffered saline
(PBS) containing 2 mM EDTA, and the membrane material was collected by centrifugation at 12,000 × g. Pelleted
membranes were solubilized in PBS in the presence of 2% (v/v) Triton
X-100 and Complete protease inhibitor mixture (Roche Molecular
Biochemicals). The lysate was cleared by centrifugation and
batch-incubated with the anti-SNAP-25-Sepharose for 2 h. Total
protein in the loading material was estimated by BCA protein
determination kit (Pierce) to be 250 mg. The beads were then washed in
a column with 30 ml of 100 mM NaCl, 2 mM EDTA,
20 mM Hepes, pH 7.0, 0.1% Triton X-100 (buffer A) followed
by 10 ml of buffer A adjusted to 0.3 M NaCl. Bound proteins
(1 mg in total) were eluted with 10 ml of buffer A adjusted to 0.6 M NaCl followed by 10 ml of 0.25 M NaCl, 0.2 mM EGTA, 0.1% Triton X-100, 20 mM glycine HCl
buffer, pH 2.5. Synaptotagmin in the 0.6 M NaCl eluate was
further purified by a heparin affinity chromatography. Brain monomeric
SNARE proteins were prepared as described previously (25). Plasmids
encoding glutathione S-transferase (GST) fusion proteins of
syntaxin 1A (amino acids 1-265), synaptobrevin 2 (amino acids 1-96),
the wild-type cytoplasmic part C2AB of synaptotagmin 1 (amino acids
96-421), mutated C2AB G374D, C2A domain (amino acids 95-265), and C2B
domain (amino acids 248-421) were described previously (17-19).
Recombinant proteins, purified on glutathione-Sepharose beads, were
released from GST, where necessary, by thrombin cleavage.
Re-assembly of SNARE and Synaptotagmin Complexes and Their
Quantification--
SNARE proteins or synaptotagmin (~2 µg each)
attached to appropriate Sepharose beads (~15 µl bed volume) were
incubated for 30 min at room temperature in 200 µl buffer A with 1-2
µM protein to be tested for binding. Beads were washed
three times by low speed centrifugation with 1 ml of buffer A, and
bound protein was eluted in sample buffer followed by SDS-PAGE in 12%
Ready gels (Bio-Rad) and Coomassie staining. For determination of the stoichiometry of binding, proteins bound to the beads were eluted into
sample buffer and separated by SDS-PAGE. Gels were stained with Sypro
Orange (Bio-Rad) according to the manufacturer's intstructions. Fluorescent protein bands were imaged using a CCD camera, and the
intensity of fluorescence was analyzed using ImageQuant software (Amersham Biosciences). To calculate the molar ratio of interacting proteins, the measured values of bound fluorescence for each band were
divided by their corresponding molecular masses. To estimate the
binding affinity of syntaxin/SNAP-25 to synaptotagmin,
glutathione-Sepharose beads containing 50 ng of GST-C2AB were incubated
for 30 min with 2 µM brain-purified SNAP-25
and increasing concentration of brain-purified syntaxin (0-1.7
µM) in a reaction volume of 100 µl. The beads were
washed three times with 1 ml of buffer A, and bound protein was eluted
into sample buffer. Bound SNAP-25 was analyzed by SDS-PAGE and Western
immunoblotting using a monoclonal anti-SNAP-25 antibody and enhanced
chemilumenescent kit West Dura (Pierce). A series of exposures of the
chemilumenescent bands were taken to ensure that saturation of the
Super RX x-ray film (Fuji Photo) was not reached. The film was scanned
on a trans-illumination densitometer, and the intensities of bands were
analyzed using the ImageQuant software. The density values were plotted
as a dose-response relationship using Prism (GraphPad Software).
 |
RESULTS |
We used a preparative anti-SNAP-25 immunoaffinity chromatography
to test an interaction of synaptotagmin with the SNAREs in the absence
of calcium in native material, namely bovine brain membranes. The brain
membrane extract was incubated with an anti-SNAP-25 antibody covalently
attached to Sepharose beads in the presence of 2 mM EDTA, a
calcium-chelating agent. Following a two-step elution, bound proteins
were analyzed by SDS-gel electrophoresis and Coomassie staining (Fig.
1, left panel). For
identification of protein bands both Western immunoblotting and mass
spectrometry were used (Fig. 1 and data not shown). The 0.6 M NaCl eluate contained synaptotagmin 1, whereas the pH 2.5 eluate contained the three SNAREs and complexin. To determine the
specificity of protein isolation on the anti-SNAP-25 column, we
performed a chromatography of the brain extract using control beads
with covalently attached bovine serum albumin. Myelin basic protein,
but not synaptotagmin nor complexin, bound to the control beads (Fig.
1, right panel). Thus, synaptotagmin and complexin are the
major proteins that specifically co-purify with the SNARE complexes, in
the absence of divalent cations.

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Fig. 1.
Synaptotagmin 1 interacts with SNARE
complexes in brain in the absence of calcium. 250 mg of Triton
X-100-solubilized brain membrane protein (Load) was
incubated with anti-SNAP-25-Sepharose beads (left panel) or
control BSA-Sepharose beads (right panel). Following a wash
in 0.3 M NaCl (Wash), synaptotagmin
(Syt) was eluted with 0.6 M NaCl, whereas
syntaxin (Syx), SNAP-25, synaptobrevin (Syb), and
complexin (Cxn) were eluted using 20 mM glycine
HCl, pH 2.5. Chromatography samples, after boiling in SDS-containing
loading buffer, were analyzed by SDS-PAGE followed by Coomassie
staining. The major band at 65 kDa was identified by MALDI-TOF mass
spectrometry as synaptotagmin 1. Western immunoblot for synaptotagmin 1 (lower panel) was performed using a monoclonal antibody
(clone 41.1) and an enhanced chemiluminescence technique. Myelin basic
protein (MBP) non-specifically interacted with both types of
Sepharose. The positions of Precision molecular weight markers
(Bio-Rad) are shown on the left.
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Several scenarios can account for the co-purification of synaptotagmin
with the SNAREs on the anti-SNAP-25 column. Synaptotagmin may interact
with monomeric SNAP-25 as suggested previously (20). Alternatively, it
may interact with SNARE complexes containing SNAP-25. Thus, we
investigated whether SNAP-25 can form stable complexes with syntaxin
and synaptobrevin. Fig. 2A
shows that SNAP-25, prebound to anti-SNAP-25-Sepharose beads, was able
to bind syntaxin and could only bind synaptobrevin when syntaxin was
present. Therefore, co-purification of synaptotagmin 1 during anti-SNAP-25 chromatography may be due to its interaction with either
SNAP-25 alone and/or the t-SNARE heterodimer and/or the full ternary SNARE complex. To distinguish between these possibilities, we prepared anti-SNAP-25-Sepharose beads carrying either SNAP-25 alone,
the t-SNARE heterodimer, or the full SNARE complex and incubated them
with brain-purified synaptotagmin 1. After washing, bound protein was
analyzed by SDS-PAGE and Coomassie staining (Fig. 2B). The
native synaptotagmin, in the absence of calcium, was able to interact
with the t-SNARE heterodimer and the ternary SNARE complex, but not
with SNAP-25 alone. We conclude that, in the case of brain-purified
proteins, the interaction of synaptotagmin with the native or
re-assembled SNARE complexes does not require calcium.

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Fig. 2.
Brain-purified synaptotagmin 1 binds the
t-SNARE heterodimer in the absence of calcium. A, beads
with immobilized SNAP-25 were mixed with input material composed of
syntaxin and synaptobrevin individually or together. After extensive
washing, proteins bound to the beads were eluted into sample buffer.
Boiled samples of input (left panel) and bound (right
panel) material were analyzed by SDS-PAGE followed by Coomassie
staining. SNAP-25 binds syntaxin alone and synaptobrevin only in the
presence of syntaxin. B, anti-SNAP-25 beads with prebound
ternary SNARE complex, SNAP-25 alone, or the t-SNARE heterodimer were
then incubated with brain purified synaptotagmin 1 for 30 min at room
temperature in 2 mM EDTA. After washing, boiled samples of
the input and bound proteins were analyzed by SDS-PAGE followed by
Coomassie staining. Synaptotagmin 1 binds the ternary SNARE complex and
the t-SNARE heterodimer in the absence of calcium.
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Since in the SNARE assembly pathway the stable t-SNARE heterodimer
exists in brain (21) and precedes the ternary SNARE complex (22), we
analyzed in more detail the calcium-independent interaction of
synaptotagmin with syntaxin and SNAP-25. First, we tested whether the
recombinant cytoplasmic part of synaptotagmin 1 (C2AB, amino acids
96-421) has the ability to bind monomeric SNAREs and/or the t-SNARE
heterodimer. In the absence of calcium, the cytoplasmic part of
synaptotagmin bound the t-SNARE heterodimer, but none of the monomeric
SNAREs (Fig. 3A). To determine
the binding efficiency we analyzed the stoichiometry of interaction
using fluorescent Sypro Orange staining, which labels proteins
quantitatively (23). The two t-SNAREs were incubated with the
immobilized cytoplasmic domain of synaptotagmin and, after washing, the
bound protein was analyzed by SDS-PAGE followed by Sypro Orange
staining. Quantification of bound dye in relation to the molecular
masses of the bound proteins yielded a molar ratio of 1:1.28:1.2 for
synaptotagmin, syntaxin, and SNAP-25, respectively (Fig.
3B). We conclude that the binding of GST-C2AB to the
t-SNAREs is approximately equimolar. We next investigated the
dependence for binding of SNAP-25 to synaptotagmin as a function of
syntaxin concentration. Syntaxin at concentrations ranging from 27 nM to 1.7 µM was added to GST-C2AB beads in
the presence of a constant concentration of SNAP-25. Syntaxin promoted
binding of SNAP-25 to synaptotagmin at concentrations above 100 nM with the half-maximal binding measured at 290 nM (Fig. 3C).

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Fig. 3.
The cytoplasmic part of
synaptotagmin 1 binds stoichiometrically and with high affinity to the
t-SNARE heterodimer. A, input consisting of either
monomeric SNAREs or the t-SNARE heterodimer (left panel) was
incubated for 30 min with immobilized GST-C2AB in the presence of 2 mM EDTA. After extensive washing the bound proteins
(right panel) were analyzed by SDS-PAGE followed
by Coomassie staining. The cytoplasmic part of synaptotagmin (GST-C2AB)
binds the t-SNARE heterodimer in the absence of calcium.
B, GST-C2AB immobilized on beads was incubated with syntaxin
and SNAP-25 in the presence of 2 mM EDTA. After washing,
the bound material was analyzed by SDS-PAGE followed by Sypro Orange
staining. Staining density was converted to molar ratios, demonstrating
stoichiometric binding of the t-SNARE heterodimer to the cytoplasmic
part of synaptotagmin. C, binding of SNAP-25 to GST-C2AB
beads as a function of syntaxin concentration. SNAP-25 (2 µM) was incubated with GST-C2AB (50 ng) immobilized on
beads in the presence of varying concentrations of syntaxin (0-1.7
µM). Bound SNAP-25 was analyzed by Western immunoblotting
and densitometric analysis. Fitting a dose-response relationship
yielded an EC50 for syntaxin-dependent SNAP-25
binding of 290 nM.
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Two versions of the synaptotagmin cytoplasmic domain were cloned, one
with a mutation of glycine to aspartic acid at residue 374 in the
second C2 domain (C2B) (19, 24). This mutation has been shown to
adversely effect folding of the C2B
-strand barrel structure (25).
Fig. 4B shows that the t-SNARE
heterodimer bound the wild-type cytoplasmic part, but not its mutated
version, suggesting that either both C2 domains or the C2B domain alone are required for this interaction. We therefore analyzed whether, in
the absence of calcium, individual C2 domains from the wild-type cytoplasmic part can be the t-SNARE heterodimer. Neither of the individual C2 domains was able to bind the t-SNARE heterodimer, but
when linked together they exhibited efficient binding (Fig. 4C).

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Fig. 4.
A requirement for both C2 domains of
synaptotagmin for interaction with the t-SNARE heterodimer. A,
domain structure of full-length native synaptotagmin and recombinant
fragments used for binding studies. B, GST-C2AB
carrying a mutation in its C2B domain does not bind the t-SNARE
heterodimer. Immobilized GST-C2AB (wild-type or G374D mutated version)
was incubated with the t-SNARE heterodimer in the absence of calcium.
The asterisk indicates a breakdown product of the mutated
GST-C2AB present on both the original beads (left panel) and
after incubation with syntaxin and SNAP-25 (right panel).
Input and bound proteins were analyzed by SDS-PAGE followed by
Coomassie staining. C, the input of t-SNARE heterodimer was
incubated with immobilized recombinant fragments of synaptotagmin
GST-C2A, GST-C2B, or GST-C2AB in 2 mM EDTA. Only the
cytoplasmic part containing both C2 domains (C2AB) was able to bind the
t-SNARE heterodimer.
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DISCUSSION |
Synaptotagmin, the proposed calcium sensor, is essential for
coupling the calcium signal to the synaptic vesicle exocytosis (6, 7).
In this study we addressed whether such coupling could be due to a
physical link between the calcium sensor and the SNARE fusion
machinery. We demonstrated by an anti-SNAP-25 immunoaffinity approach
that synaptotagmin, in the absence of calcium, can specifically
co-purify with the SNAREs from bovine brain detergent extract (Fig. 1).
This is in agreement with the previously observed calcium-independent
association of synaptotagmin with the SNARE complexes by anti-syntaxin
immunoprecipitation from rat brain (15) and by
-SNAP affinity
chromatography from bovine brain (16). Moreover, syntaxin itself was
originally identified as a protein that co-purifies in the absence of
calcium with synaptotagmin by anti-synaptotagmin immunoisolation (26). It is notable that further studies, using recombinant syntaxin, questioned an efficient calcium-independent link between synaptotagmin and syntaxin (12, 13), suggesting that either syntaxin may require
other proteins for this interaction or the bacterially expressed SNARE
fragments are deficient in some of their properties (11).
To address the molecular basis for the calcium-independent association
between the SNAREs and synaptotagmin, we used for the first time
brain-purified SNARE proteins and native synaptotagmin in parallel with
its truncated versions. Three SNARE proteins, when mixed, formed the
ternary complex that can bind synaptotagmin (Fig. 2). We further
analyzed the possible calcium-independent association of synaptotagmin
with the SNAREs in the stages preceding the ternary SNARE
complex. Syntaxin and SNAP-25 can form a stable intermediate on the
pathway from the three monomeric SNAREs to their ternary complex,
consistent with a central role for the t-SNARE heterodimer in SNARE
complex formation (Fig. 2A) (21, 22). Remarkably, the
t-SNARE heterodimer, but not monomeric SNAREs, was able to bind
synaptotagmin with high affinity and stoichiometrically in the absence
of calcium (Fig. 3). Association of syntaxin with SNAP-25 at the
release sites in the presynaptic membrane, therefore, may be an
important step for further engagement of both vesicular synaptotagmin
and synaptobrevin. As synaptobrevin is potently inhibited on synaptic
vesicle membrane (27), synaptotagmin interaction with the t-SNARE
heterodimer may precede and facilitate further engagement of
synaptobrevin into the fusion-competent SNARE complex.
A quantitive electron microscopy study of synapse morphology in the
absence of synaptotagmin revealed that synaptic vesicles are no longer
maintained in close proximity to the presynaptic membrane (28). One
attractive mechanism for this phenomenon was put forward, whereby
synaptotagmin interacts in the absence of calcium with monomeric
SNAP-25 to position the vesicles at the release sites (20). However,
this hypothesis is inconsistent with the observation that SNAP-25 is
found throughout the plasma membrane (21, 29). In addition, further
studies could only detect binding of synaptotagmin to SNAP-25 by
Western immunoblotting (14, 30), suggesting a low affinity interaction.
In our study monomeric SNAP-25 did not interact with synaptotagmin
(Figs. 2 and 3) probably because we used brain-purified protein that
may differ in its properties compared with SNAP-25 expressed in
bacteria. Importantly, genetic manipulations of both syntaxin and
SNAP-25 result in the uncoupling of calcium entry and exocytosis (14, 31), indicating that both t-SNAREs participate in linking synaptotagmin to the fusion machinery.
Finally, our data shows that both C2A and C2B domains of synaptotagmin
1 are required for the calcium-independent interaction with the t-SNARE
heterodimer (Fig. 4). Phospholipases, protein kinases, and other
enzymes contain a single copy of the C2 domain that is known to drive
their calcium-dependent translocation to cellular membranes
(32). A number of trafficking proteins, on the other hand,
e.g. DOC2, Munc13, rabphilin, the large family of
synaptotagmins and others, have a closely aligned double C2 domain
structure (33, 34). One remarkable property of these C2 domains is that
they often lack calcium binding aspartate residues and are not able to
bind phospholipid membranes in response to calcium (12, 34).
Intriguingly, knock-in studies of synaptotagmin 1 itself demonstrated
that calcium binding is dispensable for the C2A but not the C2B domain
(35-37). These observations point to a functional importance of the
double C2 domain structure that cannot solely be explained on the basis
of the calcium-dependent properties of each domain (37).
Indeed, our study provides a novel explanation for the functional
importance of the double C2 domain structure: two C2 domains of
synaptotagmin act together to bind, with high affinity and in the
absence of calcium, t-SNARE heterodimers in the target membranes.
 |
ACKNOWLEDGEMENTS |
We are grateful to Giampietro
Schiavo and Ed Chapman for synaptotagmin plasmids. We thank Sew
Peak-Chew for MALDI-TOF mass spectrometry protein identification.
 |
FOOTNOTES |
*
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.
To whom correspondence should be addressed. Tel.: 44-1223-402071;
Fax: 44-1223-402310; E-mail: baz@mrc-lmb.cam.ac.uk.
Published, JBC Papers in Press, December 20, 2002, DOI 10.1074/jbc.C200692200
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ABBREVIATIONS |
The abbreviations used are:
SNAP-25, synaptosome-associated protein of 25 kDa;
SNARE, soluble
N-ethylmaleimide-sensitive factor attachment protein
receptor;
GST, glutathione S-transferase;
PBS, phosphate-buffered saline;
BSA, bovine serum albumin;
MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight.
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