(Received for publication, May 22, 1995; and in revised form, June 29, 1995)
From the
While there is compelling evidence that the synaptic vesicle
protein synaptotagmin serves as the major Ca sensor
for regulated exocytosis, it is not known how Ca
binding initiates membrane fusion. Here we report that
Ca
increases the affinity, by approximately 2 orders
of magnitude, between synaptotagmin and syntaxin 1, a component of the
synaptic fusion apparatus. This effect is specific for divalent cations
which can stimulate exocytosis of synaptic vesicles (Ca
> Ba
, Sr
Mg
). The Ca
-dependence of the
interaction was composed of two components with EC
values
of 0.7 and 180 µM Ca
. The interaction is
mediated by the carboxyl-terminal region of syntaxin 1 (residues
194-288) and is regulated by a novel Ca
-binding
site(s) which does not require phospholipids and is not disrupted by
mutations that abolish Ca
-dependent phospholipid
binding to synaptotagmin. We propose that this interaction constitutes
an essential step in excitation-secretion coupling.
Exocytosis of synaptic vesicles is strictly controlled by
Ca ions (Katz, 1969; Augustine et al.,
1987). Presumably, Ca
ions initiate conformational
changes in proteins which ultimately catalyze membrane fusion. The
Ca
binding properties of the synaptic vesicle protein
synaptotagmin are consistent with the requirements for the exocytotic
Ca
receptor (Brose et al., 1992; reviewed by
DeBello et al.(1993), Popov and Poo(1993), Chapman and Jahn
(1994b)). Indeed, gene disruption (Geppert et al., 1994;
Broadie et al., 1994; Littleton et al., 1994; Nonet et al., 1994) and microinjection experiments (Elferink et
al., 1993; Bommert et al., 1993) have provided strong
evidence that synaptotagmin functions as the major Ca
sensor in regulated exocytosis. However, the mechanism by which
Ca
binding to synaptotagmin triggers membrane fusion
has yet to be elucidated. To address this issue, efforts have been
directed at identifying downstream 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 the adaptor protein AP-2 (Zhang et al., 1994).
Synaptotagmin also interacts with syntaxin 1, an abundant neuronal
plasma membrane protein associated with N-type Ca
channels (Bennett et al., 1992; Yoshida et al.,
1992; Sheng et al., 1994). This property suggests that
syntaxin may physically link the calcium receptor to the site of
Ca
influx.
Syntaxin has been shown recently to
form a complex with the synaptic vesicle protein synaptobrevin (VAMP)
and the synaptic plasma membrane protein SNAP-25. This complex serves
as the membrane receptor for the soluble proteins NSF ()(N-ethylmaleimide-sensitive fusion protein) and
SNAPs (soluble NSF attachment proteins), factors required for
intracellular membrane fusion. Therefore, syntaxin, SNAP-25, and
synaptobrevin have been designated as SNAREs (SNAP receptors
(Söllner et al., 1993a)). In addition,
each of the SNAREs is selectively proteolyzed by clostridial
neurotoxins, potent inhibitors of exocytosis (Schiavo et al.,
1992; Link et al., 1992; Blasi et al., 1993a, 1993b).
Furthermore, disruption of the syntaxin 1 gene in Drosophila abolishes evoked neurotransmission (Schulze et al.,
1995). Thus, the complex containing syntaxin, SNAP-25, and
synaptobrevin is thought to comprise the core of the exocytotic fusion
machine. Consequently, the interaction between syntaxin 1 and
synaptotagmin is of particular interest, since it could provide a
direct link between the Ca
-sensor and the fusion
apparatus. In the present study we have characterized the interaction
between synaptotagmin and syntaxin 1 and report that it is regulated by
Ca
ions.
Binding of recombinant proteins was also carried out by immunoprecipitation of syntaxin. Recombinant full-length syntaxin 1A and C2AB were incubated at the indicated concentrations in 20 mM Tris, pH 7.4, 150 mM NaCl, 0.5% Triton X-100 supplemented with EGTA or divalent cations for 4 h. Syntaxin 1 was immunoprecipitated by incubating the samples with affinity purified HPC-1 IgG (7 µg) for 2 h and 12 µl of protein G-Sepharose fast flow (Pharmacia) for 1 h. The immunoprecipitates were washed three times and analyzed by SDS-PAGE and immunoblotting as described above.
Figure 6:
Distinct structural determinants of
synaptotagmin underlie Ca-dependent syntaxin 1 and
phospholipid binding. A, Coomassie Blue-stained gel of
purified C2AB, C2A
B, and C2AB
fused to GST. A motif crucial
for Ca
-dependent phospholipid binding to the first
C2-domain (SDPYVK-L, residues 177-185 (Chapman and Jahn, 1994a)
was deleted from C2AB and designated C2A
B. For comparison, the
corresponding motif (amino acids 308-316) was also deleted in the
second C2-domain of C2AB and is designated C2AB
. Recombinant
proteins were prepared as described under ``Experimental
Procedures,'' immobilized using glutathione-Sepharose, and
subjected to SDS-PAGE on 10% gels. The GST-synaptotagmin fusion
proteins migrate at approximately 67 kDa; the lower molecular mass
bands are proteolytic fragments. Note: the prominent
40-kDa band
in the GST-C2A
B lane reflects the increased sensitivity
of this deletion mutant to bacterial proteases. B, disruption
of Ca
-dependent phospholipid binding to
synaptotagmin. GST-C2AB, GST-C2A
B, GST-C2AB
, and GST alone
were immobilized using glutathione-Sepharose (80 pmol/data point) and
assayed for Ca
-dependent phospholipid binding as
described (Chapman and Jahn, 1994a), using liposomes composed of a
mixture of 75% phosphatidylcholine and 25% phosphatidylserine and
labeled with [
H]phosphatidylcholine. Phospholipid
binding was measured in 50 mM Tris, pH 7.2, 100 mM NaCl with 2 mM EGTA (open bars) or 0.5 mM Ca
(solid bars). The figure shows total
phospholipid binding to the immobilized proteins (mean values from
triplicate determinations). C, disruption of
Ca
-dependent phospholipid binding does not inhibit
Ca
-dependent syntaxin 1 binding to synaptotagmin.
Ca
-dependent binding of syntaxin 1 to wild type
(C2AB) and mutant synaptotagmins (C2A
B, C2AB
) was assayed as
described in Fig. 2.
Figure 2:
Phospholipid and divalent cation
dependence for the association of synaptotagmin with syntaxin 1. Left panel, Ca-dependent binding of
synaptotagmin to syntaxin 1 does not require acidic phospholipids.
Recombinant synaptotagmin I (residues 97-421; designated C2AB)
and syntaxin 1A (full length) were prepared as described under
``Experimental Procedures.'' C2AB (0.8 µM) was
incubated in 20 mM Tris, pH 7.2, 150 mM NaCl, 0.5%
Triton X-100, supplemented with EGTA (2 mM), Ca
(0.5 mM), or Ca
(0.5 mM) plus
phospholipids (+PL; 3.7 mM phosphatidylcholine,
1.25 mM phosphatidylserine) in the presence (+) or
absence(-) of recombinant syntaxin 1 (0.8 µM).
Binding was monitored by immunoprecipitation using a purified
anti-syntaxin 1 monoclonal antibody (HPC-1) as described in
Experimental Procedures. The immunoprecipitates were separated by
SDS-PAGE and analyzed by immunoblotting. Immunoreactive bands were
visualized using
I-protein A and autoradiography. C2AB
was precipitated only in the presence of syntaxin 1. Coprecipitation
was strongly enhanced by Ca
and was independent of
negatively charged phospholipids. Right panel, divalent cation
specificity for promoting binding of synaptotagmin to syntaxin 1.
Binding of synaptotagmin to syntaxin 1 was assayed by
coimmunoprecipitation as described in legend for the left panel in the presence of EGTA (2 mM) or the following divalent
cation concentrations: Mg
alone, 2.5 mM;
Ca
, Ba
, Sr
, 0.5
mM each; Mg
(5 mM) in the presence
of Ca
(0.5 mM). Note that equal amounts of
syntaxin 1 were immunoprecipitated under all conditions. In the absence
of syntaxin 1, C2AB was not detected in the immunoprecipitates (not
shown).
To determine the effect of Ca ions on the
interaction between syntaxin and synaptotagmin, we immunoprecipitated
syntaxin 1 from synaptosomal detergent extracts, using two distinct
monoclonal antibodies directed against syntaxin 1, in the presence of
Ca
or excess Ca
chelator and
examined the precipitates for the presence of synaptotagmin. As shown
in Fig. 1(top panel), Ca
dramatically increased the level of synaptotagmin associated with
syntaxin 1. We did not observe the calcium-dependent recruitment of
additional proteins to the syntaxin immunoprecipitates by either
protein staining or immunoblot analysis using antibodies directed
against other synaptic proteins (data not shown). In addition, the
amount of synaptobrevin and SNAP-25, components of the SNARE complex,
bound to syntaxin was not affected by Ca
( Fig. 1and data not shown). Neither synaptotagmin nor syntaxin 1
was detected in control immunoprecipitations using anti-synaptophysin
antibodies (Fig. 1, left lanes).
Figure 1:
Coprecipitation of synaptotagmin with
syntaxin 1 from rat brain detergent extracts is stimulated by
Ca. Syntaxin 1 (syx) was immunoprecipitated
from rat brain detergent extracts as described under
``Experimental Procedures'' using monoclonal antibodies HPC-1
and 78.2 in the presence of EGTA (2 mM,
-Ca
) or Ca
(0.5 mM,
+Ca
). As a control, synaptophysin (syp)
was immunoprecipitated in parallel with monoclonal antibody Cl 7.3. The
immunoprecipitates were separated by SDS-PAGE and analyzed by
immunoblotting for synaptotagmin, syntaxin 1, synaptophysin, and
synaptobrevin. Immunoreactive bands were visualized using
I-protein A and autoradiography. Note that equal levels
of syntaxin 1 were precipitated by the antibodies under all conditions.
As expected, synaptophysin antibodies did not precipitate syntaxin 1 or
synaptotagmin but efficiently coprecipitated synaptobrevin with
synaptophysin, in agreement with our earlier observations (Edelmann et al., 1995).
To determine
whether the Ca-dependent coprecipitation is due to a
direct interaction between syntaxin 1 and synaptotagmin, we repeated
the syntaxin immunoprecipitations using purified recombinant proteins (Fig. 2, left panel). Full-length syntaxin 1A was mixed
with the cytoplasmic portion of synaptotagmin I (amino acids
97-421). This domain contains two repeats of a region homologous
to the C2-domains found in Ca
-dependent isoforms of
protein kinase C (Perin et al., 1990) and is designated C2AB.
As shown in Fig. 2(left panel), addition of
Ca
results in a 6-fold increase (determined by
phosphorimage analysis) in the level of C2AB which coprecipitates with
syntaxin 1. Surprisingly, this increase did not require, and was not
enhanced by, negatively charged phospholipids (Fig. 2, left
panel). Since Ca
binding to isolated
synaptotagmin requires the presence of negatively charged phospholipids
(Brose et al., 1992), these findings suggest that a novel
Ca
binding site regulates the synaptotagmin
syntaxin 1 interaction (discussed in more detail below).
We next
compared the effects of different divalent cations on the syntaxin
1-synaptotagmin interaction. As shown in Fig. 2(right
panel), Ba and Sr
ions
promoted the association of syntaxin 1 and synaptotagmin, albeit less
potently than Ca
. In contrast, Mg
was virtually inactive and, in addition, failed to antagonize the
effects of Ca
, even at high concentrations (5
mM; Fig. 2, right panel). These findings agree
with physiological studies demonstrating that Ba
and
Sr
, but not Mg
, ions can partially
substitute for Ca
in regulated exocytosis (Augustine et al., 1987).
The Ca dependence of the
interaction was determined by incubating rat brain detergent extracts,
containing native synaptotagmin, with immobilized syntaxin under
varying Ca
concentrations. Full-length syntaxin 1A
was expressed as a GST fusion protein and immobilized using
glutathione-Sepharose. After extensive washing, binding of
synaptotagmin was determined by immunoblot analysis. As shown in Fig. 3, the Ca
dependence of the
syntaxin-synaptotagmin interaction appears to be composed of two
distinct components. By curve fitting these individual components (see
legend, Fig. 3) we estimate that the EC
values for
the high and low affinity binding sites are 0.7 and 180 µM Ca
, respectively. There is evidence that at
least four calcium ions bind to the calcium sensor in a cooperative
manner to activate synaptic vesicle fusion (for example, see
Heidelberger et al.(1994)). Determining whether there is a
cooperative relationship between the Ca
concentration
and the interaction between syntaxin and synaptotagmin will require the
dissection of the high and low Ca
affinity
components.
Figure 3:
Ca dependence of
synaptotagmin-syntaxin binding. Full-length syntaxin 1 was prepared as
a GST-fusion protein and immobilized using glutathione-Sepharose. The
immobilized protein (0.5 nmol) was incubated with 1 mg of the Triton
X-100 rat brain synaptosomal extract (1 mg/ml) in the presence of 2
mM EGTA or 1 mM EGTA and sufficient Ca
to yield the indicated free Ca
concentration,
for 4 h at 4 °C. In parallel, GST alone was incubated with the
detergent extract as a control. Beads were washed three times and the
association of native synaptotagmin determined by SDS-PAGE and
immunoblot analysis using anti-synaptotagmin 1 monoclonal antibody Cl
41.1. Immunoreactive bands were visualized with
I-protein
A (upper panel). These data were quantified by phosphorimaging
and are plotted in the lower panel. Error bars represent the
standard deviation from the mean of three independent experiments. Two
distinct inflection points in the binding curve corresponding to high
and low affinity Ca
binding sites were observed.
Curves were fit to each of these two components using a logistic
equation (De Lean et al., 1978). From this analysis,
EC
values of 0.7 and 180 µM Ca
were calculated and are denoted by arrows (lower
panel). E corresponds to assays carried out in 2 mM EGTA.
The affinity of the synaptotagmin-syntaxin interaction
was determined by titrating syntaxin 1 with increasing concentrations
of C2AB in the presence of Ca or EGTA. Again, binding
was assayed by coprecipitation and immunoblot analysis (Fig. 4, left panels). In the presence of Ca
,
syntaxin 1 was saturated at low micromolar C2AB concentrations
(EC
0.5 µM). At saturation, the binding
stoichiometry approached 1:1 (Fig. 4, right panel). In
the presence of EGTA, only weak binding was observed, even at the
highest concentration of synaptotagmin tested (Fig. 4). Assuming
that the binding stoichiometry at saturation is not affected by
Ca
, we estimate that Ca
shifts the
affinity of the interaction by approximately two orders of magnitude (Fig. 4, lower panel).
Figure 4:
Concentration dependence of synaptotagmin
binding to syntaxin 1 in the presence and absence of
Ca. Left panel, a fixed amount of syntaxin 1
(0.5 µM) was incubated with increasing amounts of C2AB in
2 mM EGTA or 0.5 mM Ca
. Binding was
assayed by immunoprecipitation and immunoblot analysis as described
under ``Experimental Procedures.'' The bands immunoreactive
for synaptotagmin were quantified by phosphorimaging. These data are
plotted in the lower panel (closed circles, binding
measured in Ca
; open circles, binding
measured in EGTA). For the estimation of the EC
, (0.5
µM in Ca
), curves were fit to the data
using a logistic equation (De Lean et al., 1978). Right
panel, Coomassie Blue staining of the anti-syntaxin 1
immunoprecipitates obtained after incubating 10 µM C2AB
with or without 0.5 µM syntaxin 1 in 2 mM EGTA or
0.5 mM Ca
. Note that the staining of C2AB
and syntaxin 1 is of approximately equal intensity in the presence of
Ca
. H and L denote the heavy and
light chains, respectively, of the HPC-1 IgG used for
immunoprecipitation.
To understand in more detail
the interaction between syntaxin 1 and synaptotagmin, we investigated
the structural determinants which mediate
Ca-dependent binding. As in the Ca
titration experiments, full length and truncated forms of
syntaxin 1A were expressed as GST fusion proteins (Fig. 5A). These fusion proteins were immobilized using
glutathione-Sepharose and incubated with synaptosomal detergent
extracts in the presence of Ca
or EGTA. After
washing, binding of synaptotagmin, and, as controls, synaptobrevin and
Rab 3A, were determined by immunoblot analysis (Fig. 5B). As expected, immobilized full-length
syntaxin 1 bound to synaptotagmin in a Ca
-dependent
manner. This effect was specific for synaptotagmin, since
Ca
did not modulate the amount of synaptobrevin bound
to immobilized syntaxin 1 and did not cause other synaptic proteins (e.g. Rab 3A, Fig. 5B) to become associated
with the immobilized syntaxin. Synaptotagmin did not bind to residues
1-193 of syntaxin but bound efficiently, in a
Ca
-dependent manner, to residues 194-288 (Fig. 5B). Interestingly, the same region of syntaxin 1
also mediates its interaction with SNAP-25 (Chapman et al.,
1994), synaptobrevin (Calakos et al., 1994; Fig. 5B),
-SNAP (Hanson et al., 1995; Kee et al., 1995) and N-type Ca
-channels (Sheng et al., 1994). This region of syntaxin is composed of heptad
repeats with a high probability of forming coiled-coils (Chapman et
al., 1994; Kee et al., 1995), suggesting that at least
some of these interactions may be mediated by intermolecular coiled
coils.
Figure 5:
Residues 194-288 of syntaxin 1
mediate Ca-dependent synaptotagmin binding. A, Coomassie Blue-stained gel of full-length and truncated
forms of syntaxin 1 fused to GST. GST, full-length and truncated forms
of syntaxin 1A fused to GST, were purified using glutathione-Sepharose
and subjected to SDS-PAGE on 15% gels. Recombinant proteins were
visualized by staining with Coomassie Blue. B, identification
of the synaptotagmin binding domain of syntaxin. The GST-syntaxin 1
fusion proteins (0.5 nmol) were immobilized using glutathione-Sepharose
and incubated with Triton X-100 extract of synaptosomes (0.5 mg of
protein) in the presence of 2 mM EGTA(-) or 0.5
mM Ca
(+) for 4 h at 4 °C. Beads
were extensively washed and subjected to SDS-PAGE and immunoblot
analysis to detect synaptotagmin and, as controls, synaptobrevin and
Rab 3A. Immunoblots were visualized with
I-protein A. Total corresponds to 10 µg of the synaptosomal
extract.
It is notable that the removal of the transmembrane domain of
syntaxin (residues 266-288) resulted in decreased synaptotagmin
binding activity and also diminished the ability of syntaxin to bind
synaptobrevin (Fig. 5B) and -SNAP (Hanson et
al., 1995). In addition, insertion of the transmembrane region
into membranes is required for cleavage of syntaxin by botulinum
neurotoxin C1 (Blasi et al., 1993b). While it is unlikely that
the transmembrane domain participates in direct contacts with other
proteins, these data suggest it may be essential for oligomerization
and/or folding of syntaxin into its correct conformation.
The data
described above demonstrate that Ca regulates the
interaction between syntaxin 1 and synaptotagmin in the absence of
phospholipids. As mentioned above, this contrasts with the finding that
purified synaptotagmin binds Ca
only in the presence
of phospholipids (Brose et al., 1992), a property conferred by
the first C2-domain (Davletov and Südhof, 1993;
Chapman and Jahn, 1994a; Fukuda et al., 1994). Within this
domain, a short sequence of highly conserved residues (SDPYVK-L) has
been identified that is crucial for Ca
binding.
Deletion of, or point mutations within, this motif abolish
Ca
-dependent phospholipid binding to the isolated
first C2-domain (Davletov and Südhof, 1993; Chapman
and Jahn, 1994a). In contrast, the isolated second C2-domain does not
bind to phospholipids in a Ca
-dependent manner, even
though this domain contains the SDPYVK-L motif (Fukuda et
al., 1994). To determine the role of these motifs in
Ca
-dependent syntaxin 1 binding, we prepared mutant
synaptotagmins (Fig. 6A) which contained this deletion
in either the first (designated C2A
B) or second C2-domain
(designated C2AB
). The Ca
-dependent phospholipid
and syntaxin 1 binding properties of these mutants were then compared.
Deletion of the conserved motif within the first C2-domain abolished
Ca-dependent phospholipid binding, whereas deletion
of the corresponding motif within the second C2-domain had no effect (Fig. 6B). In contrast, binding of both deletion
mutants to syntaxin 1 was stimulated by Ca
to the
same extent as that of the wild type cytoplasmic domain of
synaptotagmin (Fig. 6C). These results clearly
demonstrate that Ca
-dependent binding of
synaptotagmin to syntaxin 1 involves structural features distinct from
those required for the Ca
-dependent interaction of
synaptotagmin with phospholipids. Therefore, Ca
regulates the synaptotagmin-syntaxin interaction via a novel
Ca
-binding site within the complex.
Recent studies have provided insights into the sequence of
events that may lead to bilayer fusion (reviewed by Rothman and
Warren(1994) and Jahn and Ferro-Novick(1994)). An early step in this
pathway includes assembly of the SNARE proteins synaptobrevin, syntaxin
1, and SNAP-25, linking the target membrane to the incoming carrier
vesicle. The assembled SNARE complex then recruits -SNAP, enabling
NSF to bind. NSF is an ATPase and upon hydrolysis of ATP dissociates
the SNARE complex, an event proposed to result in membrane fusion
(Söllner et al., 1993b).
How can the
Ca-stimulated interaction between synaptotagmin and
syntaxin 1 be integrated into this model? Söllner et al. (1993b) observed that in neuronal detergent extracts, a
small (substoichiometric) amount of synaptotagmin is associated with
syntaxin 1 which could be displaced by the addition of exogenous
-SNAP (Söllner et al., 1993b). It was
therefore suggested that synaptotagmin interacts with the SNARE complex
before
-SNAP and NSF bind and dissociate the complex. This view,
however, is difficult to reconcile with the dramatic increase in
affinity of synaptotagmin for syntaxin upon Ca
influx. Rather, we believe that in the nerve terminal the
association of synaptotagmin with syntaxin 1 occurs after dissociation
of the complex by NSF (O'Conner et al., 1994). In this
scenario, the NSF-dependent dissociation of the complex can be viewed
as an ATP-dependent priming step that is necessary but not sufficient
for membrane fusion. For exocytosis to proceed, dissociation needs to
be succeeded by the Ca
-dependent association of
synaptotagmin with syntaxin 1. Such a model would provide an
explanation for the apparent lack of ATP dependence in the final step
of exocytosis and puts the Ca
sensor, synaptotagmin,
closer to the fusion event (Hay and Martin, 1992; Bittner and Holz,
1992; Thomas et al., 1993; Neher and Zucker, 1993).
The
effect of Ca on the affinity of the
synaptotagmin-syntaxin interaction is likely to reflect a
conformational change in either one or both of these proteins. The
significance of the high and low affinity
Ca
-dependent components is currently under
investigation. However, the component which displays a low affinity for
Ca
(EC
= 180 µM) is
the first calcium-dependent interaction which corresponds to the
calcium dependence of neurotransmitter release reported in neurons
(EC
= 194 µM; Heidelberger et
al., 1994). We therefore propose that the putative conformational
changes associated with this component functions in the late acting
triggering step in exocytosis.
Note Added in Proof-Similar observations have recently been reported by Li, C., Ullrich, B., Zhang, J. Z., Anderson, R. G. W., Brose, N., and Südhof, T. C.(1995) Nature375, 594-599.