(Received for publication, September 27, 1995; and in revised form, December 20, 1995)
From the
Synaptotagmin serves as the major Ca sensor
for regulated exocytosis from neurons. While the mechanism by which
synaptotagmin regulates membrane fusion remains unknown, studies using Drosophila indicate that the molecule functions as a
multimeric complex and that its second C2 domain is essential for
efficient excitation-secretion coupling. Here we describe biochemical
data that may account for these phenomena. We report that
Ca
causes synaptotagmin to oligomerize, primarily
forming dimers, via its second C2 domain. This effect is specific for
divalent cations that can stimulate exocytosis of synaptic vesicles
(Ca
Ba
, Sr
> Mg
) and occurs with an EC
value
of 3-10 µM Ca
. In contrast, a
separate Ca
-dependent interaction between
synaptotagmin and syntaxin, a component of the fusion apparatus, occurs
with an EC
value of
100 µM Ca
and involves the synergistic action of both
C2 domains of synaptotagmin. We propose that Ca
triggers two consecutive protein-protein interactions: the
formation of synaptotagmin dimers at low Ca
concentrations followed by the association of synaptotagmin
dimers with syntaxin at higher Ca
-concentrations. Our
findings, in conjunction with physiological studies, indicate that the
Ca
-induced dimerization of synaptotagmin is important
for the efficient regulation of exocytosis by Ca
.
Rapid chemical signaling between neurons relies on the regulated
secretion of neurotransmitters stored in synaptic vesicles. In the
resting nerve terminal, a population of synaptic vesicles is tightly
bound, or docked, to the presynaptic plasma membrane. When the nerve
terminal is depolarized, Ca enters via voltage-gated
Ca
channels. High local concentrations of
Ca
then rapidly trigger the fusion of a subpopulation
of docked vesicles with the plasma membrane, resulting in the release
of neurotransmitters into the synaptic cleft.
While the mechanism(s)
by which Ca ions trigger membrane fusion remains
obscure, strong evidence indicates that the synaptic vesicle protein,
synaptotagmin I, plays an essential role in excitation-secretion
coupling. To date, nine isoforms of synaptotagmin have been cloned and
characterized (Perin et al., 1990; Geppert et al.,
1991; Mizuta et al., 1994; Hilbush and Morgan, 1994; Hudson
and Birnbaum, 1995; Li et al., 1995). The members of this
family are integral membrane proteins that span the vesicle membrane
once and possess a short amino-terminal intravesicular domain and a
large cytoplasmic domain (Perin et al., 1990, 1991). The
cytoplasmic domain contains two repeats homologous to the C2 domains
found in Ca
-dependent but not
Ca
-independent isoforms of protein kinase C,
suggesting that this conserved motif comprises a Ca
binding domain (reviewed by Nishizuka(1988)). Homologous domains
have also been identified in other proteins, at least some of which
interact with lipids in a Ca
-dependent manner (Clark et al., 1991; Stahl et al., 1988; Vogel, et
al., 1988; Shirataki et al., 1993). Subsequent studies
demonstrated that synaptotagmin indeed binds Ca
and
negatively charged phospholipids in a mutually dependent manner (Brose et al., 1992) via its first C2 domain (Davletov and
Südhof, 1993; Chapman and Jahn, 1994; Fukuda et
al., 1994).
The localization of synaptotagmin on synaptic
vesicles and its ability to bind Ca ions suggested
that the molecule may serve as a Ca
sensor in
exocytosis (Brose et al., 1992). This hypothesis has been the
subject of numerous studies. For example, the synaptotagmin gene has
been disrupted in Drosophila, Caenorhabditis elegans,
and mice (Littleton et al., 1994; DiAntonio and Schwarz, 1994;
Broadie et al., 1994; Nonet et al., 1993; Geppert et al., 1994). In all cases, perturbation of synaptotagmin
expression results in decreased excitation-secretion coupling. In fact,
disruption of the synaptotagmin I gene in mice virtually abolishes the
fast component of Ca
-dependent exocytosis, while both
spontaneous synaptic vesicle fusion and
Ca
-independent exocytosis triggered by
-latrotoxin are unaffected (Geppert et al., 1994).
Therefore, the fusion apparatus, while fully functional in the
synaptotagmin I-deficient mice, is not appropriately regulated by
Ca
. These studies suggest that synaptotagmin either
acts as the Ca
sensor for exocytosis or is essential
in some other capacity to maintain efficient excitation-secretion
coupling, for example by docking vesicles close to the site of
Ca
influx (Neher and Penner, 1994). The former
interpretation is strengthened by genetic studies in Drosophila, which demonstrated that discrete mutations in the
gene encoding synaptotagmin can affect the cooperativity of the
Ca
dependence of exocytosis (Littleton et
al., 1994). Furthermore, the phenotypes resulting from crosses
between Drosophila carrying distinct mutant synaptotagmin
alleles suggest that the molecule functions as a multimeric complex and
point to an essential function for its second C2 domain.
The precise
mechanism by which synaptotagmin functions in excitation-secretion
coupling remains ill-defined. To address this issue, efforts have been
made to identify effectors of Ca-synaptotagmin
action. Synaptotagmin was shown to form
Ca
-independent complexes with neurexins (Petrenko et al., 1991), a family of neuronal cell surface proteins, and
with the clathrin adaptor protein AP-2 (Zhang et al., 1994).
Synaptotagmin also interacts with the plasma membrane protein syntaxin
1 (Bennett et al., 1992), an essential component of the
synaptic vesicle fusion apparatus (Söllner et
al., 1993a, b; Blasi et al., 1993; Schulze et
al., 1995). Recently, we and others (Chapman et al.,
1995; Li et al., 1995) have reported that the interaction
between synaptotagmin and syntaxin is promoted by Ca
.
The Ca
dependence and divalent cation specificity of
binding were consistent with those observed for exocytosis, suggesting
that this interaction may comprise a step in transducing Ca
transients to membrane fusion.
We have continued to look for
additional effectors of Ca-synaptotagmin action and
report here that the second C2 domain of the protein is involved in the
Ca
-triggered formation of homodimers. These findings
may begin to provide the biochemical basis for the defects in synaptic
transmission imparted by mutant synaptotagmins in vivo.
The
association of recombinant regions of synaptotagmin with recombinant
syntaxin was assayed by co-immunoprecipitation using anti-syntaxin
antibodies. Recombinant full-length His-syntaxin and
His
-C2AB, or C2AB, C2A
, and C2B
generated by thrombin cleavage of their respective GST-fusion
proteins, were incubated at the indicated concentrations in 50 mM HEPES, pH 7.6, 100 mM NaCl, and 0.5% Triton X-100
supplemented with EGTA or CaCl
for 2.5 h.
His
-syntaxin was immunoprecipitated by incubating the
samples with purified HPC-1 IgG (5 µg) for 1.5 h and 12 µl of
protein G-Sepharose Fast-flow (Pharmacia Biotech Inc.) for 1 h. The
immunoprecipitates were washed three times, subjected to SDS-PAGE, and
immunoblotted, as described above, using rabbit antisera (kindly
provided by T. C. Südhof) directed against either
the first C2 domain (antisera W855; used to detect C2AB and
C2A
) or the second C2 domain of synaptotagmin (antisera
Y940; used to detect C2B
).
To determine the effect of Ca ions on the
interaction of synaptotagmin with other synaptic proteins, we
immobilized the cytoplasmic domain of the protein as a GST fusion
protein (GST-C2AB) bound to glutathione-Sepharose and used it as an
affinity matrix. When incubated with rat brain detergent extracts in
the presence of Ca
or EGTA, we observed a striking
Ca
-dependent association of native synaptotagmin with
GST-C2AB (Fig. 1). Binding was specific for synaptotagmin since
no detectable binding of other synaptic proteins, including syntaxin,
synaptophysin, SNAP-25, and synaptobrevin, was observed. To determine
the region of synaptotagmin that mediates
Ca
-dependent oligomerization, different domains of
the molecule were immobilized as GST fusion proteins and analyzed for
native synaptotagmin binding as described above. Long (with flanking
sequences; GST-C2A
) and short (without flanking sequences;
GST-C2A
) versions of the isolated first C2 domain did not
bind any of the synaptic proteins analyzed. In contrast, a long version
of the isolated second C2 domain (GST-C2B
) bound native
synaptotagmin as efficiently as the fragment containing most of the
cytoplasmic domain of the protein (GST-C2AB). This property was
preserved in the minimal second C2 domain sequence
(GST-C2B
) originally defined by its homology to protein
kinase C (Perin et al.(1990); Fig. 1). Therefore, the
second C2 domain mediates Ca
-dependent
self-association of synaptotagmin.
Figure 1:
Ca-induced
self-association of synaptotagmin is mediated through the second C2
domain. Domains of synaptotagmin (upper panel) fused to GST
(0.5 nmol), were immobilized using glutathione-Sepharose and incubated
for 2.5 h with rat brain detergent extracts in 2 mM EGTA
(-Ca
) or 1 mM Ca
(+Ca
). Beads were washed with binding
buffer, and bound proteins were analyzed by SDS-PAGE and immunoblotting
using
I-protein A as described (Chapman et al.,
1995). One-tenth of the samples were analyzed. For comparison, 4 µg
of total rat brain detergent extract were analyzed in parallel (total). Native synaptotagmin was detected using an antibody
directed against the luminal domain (604.1; Chapman and Jahn(1994)).
The amino acid residues of each recombinant synaptotagmin construct are
listed in the upper panel (right). TMR,
transmembrane region. Boxed regions indicate sequence
conservation with the C2 domains of protein kinase C, as defined by
Perin et al.(1990). The C2 domain closest to the transmembrane
segment is designated A; the more distal, carboxyl-terminal C2
domain, is designated B.
It has been reported previously
that the first C2 domain of synaptotagmin mediates the
Ca-dependent component of phospholipid binding by the
molecule (Fukuda et al., 1994; Chapman et al., 1995),
while the second C2 domain specifically mediates
Ca
-independent binding to AP-2 and IP
(Zhang et al., 1994; Fukuda et al., 1994). Our
finding that C2B mediates Ca
-dependent
oligomerization demonstrates yet another functionally divergent
property of the individual C2 domains of synaptotagmin and represents
the first Ca
-dependent interaction that directly
involves the second C2 domain of the protein.
We and others have
recently demonstrated that synaptotagmin binds to syntaxin in a
Ca-dependent manner (Chapman et al., 1995;
Li et al., 1995). Therefore, the failure of native syntaxin to
bind to the immobilized GST-C2AB shown in Fig. 1was surprising.
However, when C2AB is removed from the GST-glutathione-Sepharose by
thrombin cleavage it then associates with immunoprecipitated syntaxin
in a Ca
-dependent manner (Fig. 2).
Ca
stimulated the binding of C2AB to syntaxin
6-8-fold, in agreement with our previous observations
(Chapman et al., 1995). This effect is not due to the
Ca
-dependent oligomerization of C2AB associated with
syntaxin since recombinant C2AB only weakly oligomerizes in a
Ca
-dependent manner (described in detail below).
Therefore, fusion of C2AB to GST and/or its immobilization onto
glutathione-Sepharose diminishes its ability to bind syntaxin in a
Ca
-dependent manner. To further compare
Ca
-dependent synaptotagmin oligomerization and
syntaxin binding, we examined the abilities of the isolated C2 domains,
C2A
and C2B
, to co-immunoprecipitate with
syntaxin in the presence and absence of Ca
. As shown
in Fig. 2, under the conditions of our assay, C2A
did not co-immunoprecipitate with syntaxin either in the presence
or absence of Ca
. Ca
-independent
binding of C2B
to syntaxin was observed, which was only
weakly enhanced (
1.5-fold) by Ca
. These results
indicate that both C2 domains act in a synergistic manner to confer
Ca
-dependent synaptotagmin binding to syntaxin.
Figure 2:
Ca-dependent
synaptotagmin-syntaxin binding involves the synergistic action of both
C2 domains of synaptotagmin. Soluble forms of C2AB, C2A
,
and C2B
were prepared by thrombin cleavage of the
GST-fusion proteins described in Fig. 1(Chapman et
al., 1995). Ca
-dependent binding of these
fragments (0.5 µM) to His
-syntaxin (0.7
µM) was analyzed by co-immunoprecipitation using
anti-syntaxin antibodies in the presence of 2 mM EGTA or 0.5
mM Ca
, as described under
``Experimental Procedures.'' Bound recombinant synaptotagmins
were detected using rabbit antisera directed against the individual C2
domains of the protein. Immunoreactive bands were visualized using
enhanced chemiluminescence (Amersham Corp.). One-tenth of the
immunoprecipitates were analyzed for recombinant synaptotagmin binding. total corresponds to of the starting
material.
Our data demonstrating that the isolated C2A region of synaptotagmin
does not form a stable complex with syntaxin do not agree with the
findings of Li et al.(1995), who reported a
Ca-dependent interaction between immobilized
GST-C2A
and native syntaxin. We observed low levels of
Ca
-dependent syntaxin binding to immobilized
GST-C2A
only after overnight incubation periods or by using
very high Ca
concentrations (i.e. 2.5 mM Ca
). These findings, in conjunction with the
data described above, indicate that both C2 domains of synaptotagmin
are required for efficient binding to syntaxin.
We next tested the
ability of different divalent cations to stimulate synaptotagmin
oligomerization. Binding assays using immobilized GST-C2AB and rat
brain detergent extracts were carried out as described in Fig. 1in the presence of EGTA, Mg,
Ca
, Ba
, or Sr
.
Self-association of synaptotagmin was specifically promoted by
Ca
ions (Fig. 3). These data agree with
physiological studies demonstrating that Ca
selectively triggers exocytosis (reviewed by Augustine et
al.(1987)). The inability of Sr
to promote
oligomerization is of particular interest since this divalent cation is
unable to support the fast component of Ca
-dependent
exocytosis (Goda and Stevens, 1994). This finding is consistent with
the notion that the Ca
-dependent oligomerization of
synaptotagmin may be an essential step in mediating this rapid
component.
Figure 3:
Ca selectively promotes
synaptotagmin oligomerization. Binding of native synaptotagmin to
GST-C2AB was assayed as described in Fig. 1with the following
additions: 2 mM EGTA, 0.5 mM Ca
,
2.5 mM Mg
, 0.5 mM Sr
, or 0.5 mM Ba
. The
Mg
sample also contained 2 mM EGTA. Binding
was detected by SDS-PAGE and immunoblotting as described in Fig. 1.
In parallel experiments, we found that recombinant
His-C2AB also readily binds to immobilized GST-C2AB.
However, this interaction was weakly (
2-fold) and inconsistently
stimulated by Ca
(data not shown). The more tightly
regulated Ca
-dependent interaction between native
synaptotagmin derived from rat brain detergent extracts and GST-C2AB
does not appear to be due to other factors (i.e. proteins or
lipids) present in the extracts since the addition of the extract to
the binding assay mixture does not restore Ca
responsiveness to recombinant C2AB (data not shown). We also
addressed the possibility that residues 1-96 of synaptotagmin,
which are not present in C2AB, are essential for
Ca
-dependent synaptotagmin oligomerization. However,
full-length synaptotagmin derived from transfected COS-7 cells or
generated by in vitro transcription and translation also
failed to consistently exhibit significant
Ca
-dependent GST-C2AB binding (data not shown).
However, these full-length forms of synaptotagmin have greater
electrophoretic mobilities than the native protein, suggesting that
they are not properly processed or folded. In summary,
Ca
-dependent oligomerization of synaptotagmin can
only be measured when one of the binding partners is brain-derived
native protein.
We therefore investigated the Ca dependence of synaptotagmin oligomerization by monitoring the
binding of native synaptotagmin, derived from brain extracts, to
immobilized GST-C2AB (Fig. 4, upper panel). Bound
synaptotagmin was analyzed by immunoblotting as described and
quantitated by PhosphorImager analysis, yielding an EC
value of 3-10 µM Ca
(Fig. 4, bottom panel, closed circles).
Figure 4:
Synaptotagmin oligomerization and syntaxin
binding have distinct Ca dependences. Upper
panel, the association of native synaptotagmin with GST-C2AB was
assayed as described in Fig. 1. Samples were buffered with 2
mM EGTA (EGTA) or 1 mM EGTA plus
Ca
to yield the indicated free Ca
concentration. Middle panel, the association of
His
-C2AB (0.4 µM) with
His
-syntaxin (0.7 µM) was assayed by
co-immunoprecipitation using anti-syntaxin antibodies as described
under ``Experimental Procedures.'' Samples were buffered with
2 mM EGTA (EGTA) or 1 mM EGTA plus
Ca
to yield the indicated free Ca
concentration. Both His
constructs contain the T7 tag
and were visualized using a T7 tag monoclonal antibody (Novagen) and
I-protein A. H and L denote the heavy
and light chains of the anti-syntaxin antibody used for
immunoprecipitation. Bottom panel, data were quantified by
PhosphorImager analysis, and the percentage of maximal binding was
plotted versus the free Ca
concentration. Closed circles represent native synaptotagmin binding to
GST-C2AB; open circles represent His
-C2AB binding
to His
-syntaxin. E, 2 mM EGTA.
In a previous study we observed that the Ca dependence for the association of native synaptotagmin with
immobilized syntaxin was composed of two components (Chapman et
al., 1995). The minor component required Ca
concentrations in the low micromolar range (EC
1 µM Ca
), very similar to
Ca
dependence reported here for the oligomerization
of synaptotagmin (EC
= 3-10 µM Ca
). Thus it is possible that this minor
component reflected the Ca
-dependent oligomerization
of native synaptotagmin associated with the immobilized syntaxin rather
than being due to a direct increase in binding between the two
different proteins. If this assumption is correct, only the second (and
major) binding component, with an EC
value of
approximately 180 µM Ca
, would reflect
the direct association of native synaptotagmin with the immobilized
recombinant syntaxin. To address this possibility, we reexamined the
interaction of synaptotagmin with syntaxin using recombinant
His
-C2AB, which does not exhibit significant
Ca
-dependent oligomerization. In principle, the
Ca
dependence for the interaction of
His
-C2AB with syntaxin should be composed of a single
component without a significant contribution due to
His
-C2AB oligomerization. We mixed purified
His
-C2AB with purified His
-syntaxin at
different Ca
concentrations and immunoprecipitated
the resultant complexes with monoclonal antibodies directed against
syntaxin. As expected (Fig. 4, middle and lower
panels, open circles), the Ca
dependence for binding of His
-C2AB to
His
-syntaxin was composed of a single component with an
EC
value of
100 µM Ca
.
This value is in reasonable agreement with the previously reported
value of 180 µM Ca
described above.
Therefore, synaptotagmin oligomerization (EC
=
3-10 µM Ca
) and syntaxin binding
(EC
100 µM Ca
) are
clearly distinct Ca
-dependent interactions.
Finally, we addressed the stoichiometry of the
Ca-induced synaptotagmin oligomers using sucrose
density gradient centrifugation. Previous reports had shown that
synaptotagmin was broadly distributed on density gradients when
solubilized in CHAPS or Zwittergent 3-10 (Brose et al.,
1992; Perin et al., 1991). Using Triton X-100-solubilized rat
brain membranes, Garcia et al. (1995) reported a more compact
distribution, with a major peak corresponding to the dimeric form of
synaptotagmin (
100 kDa). It is notable that in their study (Garcia et al., 1995), the free Ca
was unbuffered
and can therefore be assumed to be present at µM levels.
We therefore examined the effect of Ca
and EGTA on
the migration of native synaptotagmin on similar gradients, using
Triton X-100-solubilized rat brain membranes as a protein source. As
shown in Fig. 5, synaptotagmin peaks at a position corresponding
to its monomeric molecular mass in EGTA (
50 kDa) but exhibits a
dramatic shift in the presence of Ca
, peaking at a
position corresponding to the molecular mass of its dimeric form
(
100 kDa). This shift was highly specific for synaptotagmin; no
shift was observed for synaptophysin, rab 3A (Fig. 5), or other
synaptic proteins, including the Ca
and phospholipid
binding protein rabphilin (data not shown). Under these dilute
conditions, little of the total synaptotagmin is bound to syntaxin
(<10% in Ca
, as determined by immunoprecipitation
of syntaxin), and as expected, syntaxin did not shift in the gradient (Fig. 5). Since this approach does not rely on the use of
recombinant proteins, it demonstrates that Ca
can
induce the oligomerization of native synaptotagmin.
Figure 5:
Ca induces the formation
of native synaptotagmin dimers. Rat brain Triton X-100 extracts were
prepared as described under ``Experimental Procedures'' and
supplemented with either 2 mM EGTA or 1 mM Ca
. Five hundred-µl aliquots were subjected
to density gradient centrifugation on 5-15% sucrose gradients.
Aliquots from 12 0.5-ml fractions corresponding to the top of the
gradient were analyzed by SDS-PAGE and immunoblotting using monoclonal
antibodies directed against the indicated proteins and visualized by
enhanced chemiluminescence (Amersham). The intensities of the
synaptotagmin immunoreactive bands were determined by densitometry and
are plotted in the bottom panel (closed circles,
EGTA; open circles, Ca
). In the presence of
Ca
, the peak of synaptotagmin immunoreactivity shifts
from
50 to
100 kDa. Molecular mass markers were as follows:
29 kDa (carbonic anhydrase), fraction 4; 45 kDa (ovalbumin), fraction
5; 67 kDa (bovine serum albumin), fraction 6; 150 kDa (alcohol
dehydrogenase), fraction 9.
In order to understand how the Ca-binding
protein synaptotagmin functions in regulated exocytosis it is essential
to identify and characterize effectors for
Ca
-synaptotagmin action. Previous work has shown that
these effectors include negatively charged phospholipids (Brose et
al., 1992; Davletov and Südhof, 1993; Chapman
and Jahn, 1994) and syntaxin (Chapman et al., 1995; Li et
al., 1995). Here we report that Ca
induces the
formation of synaptotagmin dimers. Domain mapping analysis demonstrated
that the second C2 domain (C2B) is involved in
Ca
-dependent oligomerization, while both C2 domains
are required for efficient Ca
-dependent
synaptotagmin-syntaxin binding. Interestingly, the second C2 domain of
synaptotagmin is essential for efficient Ca
-regulated
exocytosis in Drosophila (Littleton et al., 1994).
Few larvae with a mutant allele lacking the second C2 domain are
viable. Of the surviving larvae, the cooperativity of
excitation-secretion coupling is reduced 2-fold. In addition, an allele
carrying a point mutation in the second C2 domain confers a higher
requirement for extracellular Ca
, while the
cooperativity of release remains comparable with that observed in wild
type flies. These studies suggest that the Ca
-induced
formation of synaptotagmin dimers via the second C2 domain is essential
for efficient excitation-secretion coupling.
We have determined the
Ca dependences for the oligomerization of
synaptotagmin and the interaction of synaptotagmin with syntaxin and
found that they differ by an order of magnitude. These findings suggest
a sequential order of events, which are summarized in the model shown
in Fig. 6. As Ca
levels rise from resting (0.1
µM) to low µM levels (3-10
µM), synaptotagmin forms dimers via its second C2 domain.
As Ca
levels rise to concentrations capable of
triggering exocytosis (20-200 µM; Heidelberger et al.(1994)), synaptotagmin dimers bind to syntaxin via the
synergistic action of both C2 domains.
Figure 6:
Model depicting the sequential effects of
Ca on synaptotagmin-effector interactions. Light
shading denotes the first C2 domain of synaptotagmin (C2A), darker
shading indicates the second C2 domain (C2B); syntaxin is shown in black. Arrows in the right panel denote the
Ca
-dependent interaction of C2A and the
Ca
-independent interaction of C2B of synaptotagmin
with negatively charged phospholipids in either the synaptic vesicle or
the plasma membrane.
As described above, nine
isoforms of synaptotagmin have been identified thus far. The findings
presented in this study raise the possibility that low levels of
Ca may trigger the association of different
synaptotagmin isoforms with one another. Future experiments will be
directed at determining whether such complexes form and whether these
complexes exhibit distinct Ca
-dependent interactions
with downstream effectors.