Calcium-dependent Oligomerization of Synaptotagmins I
and II
SYNAPTOTAGMINS I AND II ARE LOCALIZED ON THE SAME SYNAPTIC
VESICLE AND HETERODIMERIZE IN THE PRESENCE OF CALCIUM*
Shona L.
Osborne,
Judit
Herreros,
Philippe I. H.
Bastiaens
, and
Giampietro
Schiavo§
From the Molecular Neuropathobiology and
Cell
Biophysics Laboratories, Imperial Cancer Research Fund, 44 Lincoln's
Inn Fields, London WC2A 3PX, United Kingdom
 |
ABSTRACT |
Synaptotagmins constitute a large family of
membrane proteins characterized by their distinct distributions and
different biochemical features. Genetic evidence suggests that members
of this protein family are likely to function as calcium sensors in
calcium-regulated events in neurons, although the precise molecular mechanism remains ill defined. Here we demonstrate that different synaptotagmin isoforms (Syt I, II, and IV) are present in the same
synaptic vesicle population from rat brain cortex. In addition, Syt I
and II co-localize on the same small synaptic vesicle (SSV), and they
heterodimerize in the presence of calcium with a concentration dependence resembling that of the starting phase of SSV exocytosis (EC50 = 6 ± 4 µM). The
association between Syt I and Syt II was demonstrated by
immunoprecipitation of the native proteins and the recombinant
cytoplasmic domains and by using fluorescence resonance energy transfer
(FRET). Although a subpopulation of SSV containing Syt I and IV can be
isolated, these two isoforms do not show a
calcium-dependent interaction. These results suggest that
the self-association of synaptotagmins with different calcium binding
features may create a variety of calcium sensors characterized by
distinct calcium sensitivities. This combinatorial hypothesis predicts
that the probability of a single SSV exocytic event is determined, in
addition to the gating properties of the presynaptic calcium channels,
by the repertoire and relative abundance of distinct synaptotagmin
isoforms present on the SSV surface.
 |
INTRODUCTION |
Neuronal communication depends upon the transduction of an
electric nerve impulse for the release of neurotransmitters from their
storage compartment, the small synaptic vesicles
(SSV).1 This tightly
regulated process is triggered by the rapid increase of the
intracellular calcium due to the opening of voltage-gated calcium
channels at the active zones of the synaptic plasma membrane, which
causes the activation of the SSV fusion machinery (1, 2). Of the
proteins involved in the physiological cycle of the SSV, the members of
the synaptotagmin family represent the best candidates for the role of
calcium sensors in neurotransmitter release and, more generally, in
regulated exocytosis (3-5). This protein family comprises more than a
dozen members having the same overall structure and a variable degree
of homology (4, 5). Synaptotagmins are integral membrane proteins with
broad distributions in neuronal and non-neuronal tissues that vary
between isoforms (4, 5). In the central nervous system and
neuroendocrine cells, synaptotagmins are localized on SSV and secretory
granules (6). These proteins are characterized by a single
membrane-spanning domain and by a large cytoplasmic portion containing
two internal repeats that have homology with the C2 domain of protein
kinase C (7, 8). This domain is known to regulate the
calcium-dependent translocation of protein kinase C to
membranes and is present in many proteins with different functions (5,
8). In synaptotagmins, the two C2 domains are responsible for different
interactions, both calcium-dependent and
calcium-independent. The first C2 homology domain of synaptotagmin,
termed C2A, whose structure has been recently solved (9-12), binds
calcium and acidic phospholipids (in particular, phosphatidylserine) in
a ternary complex in a calcium-dependent manner
(EC50 = 3-6 µM Ca2+) (13-16).
The C2A also binds syntaxin, a presynaptic t-SNARE (17) at relatively
high calcium concentrations (EC50 > 200 µM
Ca2+) (11, 18, 19). The second C2 domain (C2B) of
synaptotagmin I (Syt I) has recently been shown to interact with
another class of acidic phospholipids, the phosphoinositides. The
specificity of this interaction is strictly dependent on calcium as
illustrated by the maximal binding of phosphatidylinositol
4,5-diphosphate at calcium concentrations between 20 and 100 µM (20). The C2B domain also shows calcium-independent
interactions. Among these, its binding to the soluble phosphoinositide
analogues, inositol polyphosphates, may be important in modulating the
interaction of synaptotagmin with phosphoinositides (21-23). Other
interactions independent of calcium are with the clathrin adaptor AP-2
(24) and with
-SNAP, the neuronal isoform of a family of cytosolic proteins termed soluble N-ethylmaleimide-sensitive factor
(NSF) attachment proteins (SNAPs) (25). Synaptotagmin is also able to
bind the other synaptic t-SNARE, SNAP-25. While the interaction with
syntaxin is totally calcium-dependent, SNAP-25 binding to synaptotagmin is only partially modulated by calcium (26-28).
Synaptotagmin also interacts with voltage-gated calcium channels
(29-38). These findings, together with functional studies (39-41) and
genetic evidence from Caenorhabditis elegans,
Drosophila, and mouse (42-45) strongly indicate that
synaptotagmins are the main calcium sensor(s) for synchronous
neurotransmitter release in the central nervous system.
Recently, another protein-protein interaction involving the C2B domain
of synaptotagmin has been described. This interaction involves the
calcium-dependent self-association of the C2B domain of Syt
I and consequent synaptotagmin dimerization (46, 47). This result has
suggested a model in which, following calcium rise at the nerve
terminal, synaptotagmin first dimerizes and then associates with
syntaxin, creating a protein complex involved in the
calcium-dependent step(s) leading to SSV fusion with the active zones of the synaptic plasma membrane (47).
In this paper, we present evidence for a broader distribution of some
synaptotagmin isoforms than expected on the basis of the reported
literature. We analyzed SSV preparations isolated from adult rat brain
cortex for the presence of synaptotagmins I, II, and IV (Syt I, Syt II,
and Syt IV) and found that the same population of SSV contains an
overlapping distribution of these isoforms. In addition, both Syt I and
II are present on the same SSV, and they heterodimerize efficiently
with a concentration dependence for calcium resembling that of the
starting phase of SSV exocytosis. This process was confirmed in
vitro with the soluble cytoplasmic domains of Syt I and II using
both immunoprecipitation and fluorescence resonance energy transfer
(FRET) techniques. In contrast, the distribution of Syt IV on SSV only
partially overlaps with that of Syt I and II, and the ability of Syt IV to associate with Syt I is low. Taken together, these results suggest
that the self-association of synaptotagmins with different calcium
binding features possesses a certain degree of specificity and may
create a variety of calcium sensors characterized by distinct calcium sensitivities.
 |
EXPERIMENTAL PROCEDURES |
Antibody Production and Purification--
Peptides
MVSASHPEALAAPVTTVATC (corresponding to residues 1-19 of rat Syt I with
an additional cysteine at the C terminus), CMRNIFKRNQEPIVAPAT (residues
1-17 of rat Syt II), and CMAPITTSRVEFDEIPT (residues 1-16 of rat Syt
IV; both peptides with an additional cysteine at the N terminus) were
conjugated to maleimide-activated keyhole limpet hemocyanin (Pierce)
following the manufacturer's specifications. Polyclonal
isoform-specific antibodies were raised in New Zealand White rabbits by
intramuscular injection of 1 mg of the corresponding peptide together
with Freund's adjuvant. The antibodies were affinity-purified by using
Sulfolink resin (3 ml; Pierce) previously coupled with 3 mg of the
specific peptide and eluted with 100 mM glycine, pH 2.5, 0.1% bovine serum albumin. This method yielded a satisfactory
purification of anti-Syt I and -Syt II antibodies, but not of anti-Syt
IV, that either proved sensitive to both acidic and basic elution
protocols or did not elute from the peptide column (48). Anti-Syt-IV
antibodies were partially purified by ammonium sulfate precipitation
and protein A-Sepharose affinity chromatography. The specificity of the
purified antibodies was evaluated by neutralization with the generating peptides and by comparison with the preimmune serum.
Synaptic Vesicle Purification and Immunoisolation--
SSV from
rat brain cortex were prepared following the method of Huttner et
al. (49) with minor variations. Briefly, the membrane-enriched
fraction (LP2) obtained by centrifugation of the lysate supernatant was
resuspended as described and then loaded into a linear continuous
sucrose gradient prepared from equal amounts of 50 and 800 mM sucrose (Life Technologies, Inc.) by using a model 106 Gradient Master (BioComp Instrument Inc., Fredericton, Canada). Samples
were spun at 26,000 rpm (90,000 × gav) in
a Beckman SW28 rotor for 4 h. The fraction corresponding to a
sucrose concentration range between 200 and 400 mM and
visible as a translucent band was loaded into a glycerol-coated
controlled pore glass beads column (25 × 1000 mm; CPG, Lincoln
Park, NJ) equilibrated in 4 mM HEPES-NaOH, pH 7.4, 300 mM glycine, 0.04% NaN3. The column was eluted
at 0.7 ml/min with the same buffer, and 8-ml fractions were collected.
For the data presented in Fig. 1B, proteins corresponding to
each fraction (400 µl) were recovered by precipitation with trichloroacetic acid by using 0.5 µg of sodium deoxycholate as carrier and then analyzed by SDS-PAGE. For immunoprecipitation and
immunoisolation experiments, fractions corresponding to the second peak
of absorbance at 280 nm were pooled and centrifuged at 45,200 rpm
(198,000 × gav) in a 50.2 Ti rotor for
2 h. The SSV pellet was resuspended in 1 ml of 4 mM
HEPES-NaOH, pH 7.4, 300 mM glycine, 0.04%
NaN3, 100 µM phenylmethylsulfonyl fluoride, 2 µg/ml pepstatin, and the suspension was homogenized by forcing it
eight times through a 25-gauge needle. Protein concentration was
routinely determined with Bio-Rad protein assay reagent by using
purified immunoglobulins as a reference.
For SSV immunoisolation purposes, M48 monoclonal antibodies against Syt
I (50) were purified with DEAE blue resin (Bio-Rad) (51) and then
dialyzed extensively against distilled water. The purified IgG fraction
(0.4 mg) was coupled to Eupergit C1Z methacrylate microbeads (98.6 mg;
1-µm diameter; Röhm Pharma, Darmstadt), previously washed with
distilled water, by incubation at room temperature for 1 h (52).
The reaction was stopped by centrifugation, followed by incubation with
1 M glycine, pH 6.5, for 12 h at room temperature to
block the remaining reactive sites. Beads were subjected to six
alternate pH washes (100 mM Tris-HCl, pH 8.0, 150 mM NaCl and 100 mM sodium acetate, pH 4.5, 150 mM NaCl) to remove loosely bound material. The beads were
stored in 20 mM Tris-HCl, pH 7.2, 150 mM NaCl,
0.02% NaN3. In selected experiments, protein G-Sepharose
fast flow beads (Amersham Pharmacia Biotech) previously conjugated with
M48 monoclonal antibodies (50) were used. Immunoisolation was performed
by using the purified SSV fraction obtained from the glycerol-coated
controlled pore glass beads column as starting material. 10 µl of
50% M48-conjugated and control beads were washed in glycine buffer (4 mM HEPES-NaOH, pH 7.4, 300 mM glycine, 0.04%
NaN3) and preincubated with 1 mg of asolectin (Sigma) to
block any nonspecific lipid binding sites. They were washed again in
glycine buffer and incubated with 10 µg of SSV in the presence of 0.2 mg/ml ovalbumin, 5% glycerol at 4 °C for 2 h. The reaction was
stopped by centrifugation. The beads were washed three times in glycine
buffer and resuspended in SDS-containing sample buffer, while the
supernatant proteins were precipitated with trichloroacetic acid in the
presence of 0.05% (w/v) sodium deoxycholate as carrier and resuspended
as above. Proteins were analyzed by SDS-PAGE and Western blotting using
anti-Syt II, anti-Syt IV, and anti-VAMP 2 (WAKO, Richmond, VA)
antibodies. Immunoreactive bands were visualized with a rabbit anti-mouse IgG or a sheep anti-rabbit peroxidase conjugated IgG followed by ECL detection (Amersham Pharmacia Biotech).
Immunoprecipitation of Native Synaptotagmins--
50 µg of the
column-purified pooled cortical SSV fraction and 100 µg of an impure
vesicular fraction (LP2) from rat cerebellum were solubilized in 25 mM HEPES-KOH, pH 7.6, 100 mM KCl, 1% glycerol containing 100 µM phenylmethylsulfonyl fluoride, 2 µg/ml pepstatin, and 4% octyl-
-D-glucosopyranoside
(OG) for 30 min at 4 °C. Detergent-free buffer was added to give a
final OG concentration of 1.2%, and the solutions were centrifuged to
remove insoluble material. The solubilized proteins were incubated for
2 h at 4 °C with protein G-agarose beads (Boehringer Mannheim)
previously conjugated with M48 monoclonal antibodies (50) or anti-Syt I
or anti-Syt II antibodies or with the protein G beads alone. The
reactions were stopped by centrifugation. The proteins in the
supernatant were precipitated with trichloroacetic acid and resuspended
in 20 µl of SDS-containing sample buffer, while the beads were washed
three times in incubation buffer containing 0.8% OG and then prepared for SDS-PAGE. 8 µl of each sample was analyzed by SDS-PAGE and Western blotting, using anti-Syt I, anti-Syt II or anti-Syt IV antibodies.
For the experiments investigating the calcium dependence, similar
procedures were used, except that Ca2+/EGTA (final
concentration 2 mM EGTA) buffers were added to the incubation buffer to yield the free calcium concentrations indicated. 1 mM MgCl2 was also added, and the
immunoprecipitation step was carried out in 2% OG. Immunoblots were
detected either with ECL or by incubation with iodinated anti-rabbit
secondary antibodies (17.6 µCi/µg, 1 µCi/ml, Amersham Pharmacia
Biotech). In the case of Western blotting detected with the ECL
detection system, Syt II and Syt IV were quantified with the NIH Image
(version 1.61) software by comparing the signal present in the
immunoprecipitate with that obtained from an antigen standard curve
prepared by loading increasing amounts of either Syt II or Syt IV
(0.25, 0.5, 0.75, and 1 times the starting material) (53). Quantitation of Syt II with iodinated anti-rabbit secondary was performed by using a
Molecular Dynamics PhosphorImager. Syt recovery in the immunoprecipitate was calculated as the percentage of total Syt II
present in the sample. Both the sum of the immunoreactivity present in
the supernatant (S) and in the pellet (P) and the total input of the
sample (T) were used as denominators to calculate the percentage of Syt
recovery, and the two values were compared. In all cases, the maximal
S.D. between the measurements obtained with the two methods was less
than 10, with an average value of 6. Both values were used to determine
the EC50 of the calcium dependence of Syt I/Syt II
co-immunoprecipitation, and the variability between the two methods is
taken into account in the error bars in Fig. 4.
To compare different experiments, the percentage of Syt II present in
the Syt I immunoprecipitate was expressed as a percentage of the
maximal recovery (Fig. 4).
Expression and Immunoprecipitation of Recombinant
Synaptotagmins--
Recombinant glutathione S-transferase
(GST)-synaptotagmin fusion proteins were prepared by inserting the DNA
corresponding to residues 95-421 of rat Syt I and 103-422 of rat Syt
II (GenBankTM accession numbers X52772 and M64488,
respectively) into the EcoRI/NcoI sites of the
expression vector pGEX-KG (54). The proteins correspond to the
published sequences (55, 56), except for the substitution of
Glu188 for Asp, Gly374 for Asp, and
Ile393 for Met in rat Syt I. These variations, generated by
single base changes, may be ascribed to the different DNA sources used
for the cloning of rat Syt I (Rattus norvegicus here
versus Rattus rattus used in Ref. 56). For
immunoprecipitation purposes, a tagged version of the rat Syt II GST
fusion protein was prepared by inserting the sequence YPYDVPDYA
(corresponding to the hemagglutinin epitope (HA)) immediately
downstream of the thrombin cleavage site. GST fusion proteins were
purified on glutathione-agarose beads (Sigma), and the cytoplasmic
domains of Syt I and Syt II were released by thrombin cleavage (54).
Proteins were purified by ion exchange chromatography on a Mono-Q
matrix (Amersham Pharmacia Biotech), dialyzed against 20 mM
HEPES-KOH, pH 7.6, 150 mM KCl, 10% glycerol, 0.1 mM dithiothreitol, and, after freezing in liquid nitrogen,
stored at
80 °C.
For immunoprecipitation experiments, Syt I and HA-tagged Syt II were
thawed and immediately diluted in 25 mM HEPES-KOH, pH 7.5, KCl 100 mM, glycerol 1%, dithiothreitol 0.1 mM, OG 0.8% (final concentrations: Syt II 44 nM, Syt I 435 nM). The protein solution was
precleared by centrifugation to eliminate any aggregated material and
then mixed with the appropriate Ca2+/EGTA buffers to yield
the free calcium concentrations indicated. In selected samples,
MgCl2 was added to reach a free Mg2+
concentration of 0.5 mM. Samples were incubated for 1 h at 4 °C and then immunoprecipitated by adding an excess of
monoclonal anti-HA antibody 12CA5 (57) prebound to protein G-Sepharose fast flow (Amersham Pharmacia Biotech) for 1 h at 4 °C. Beads were recovered by gentle centrifugation and washed extensively with
incubation buffer containing the appropriate free calcium concentration
and 0.5 mM MgCl2 where indicated. Proteins
bound to beads were then separated by SDS-PAGE and analyzed either by densitometric analysis of the Coomassie Blue-stained gels or by Western
blotting. In both cases, Syt I recovery was quantified with the NIH
Image software by comparing the signal present in the immunoprecipitate
with that obtained from a standard curve prepared by loading increasing
amounts of recombinant Syt I (50, 100, 150, 200, and 250 ng in the case
of Western blotting and 0.2, 0.4, 0.6, 0.8, and 1 µg for Coomassie
staining) (53). Data were expressed as a percentage of the maximal Syt
I present in the Syt II immunoprecipitate.
Similar results were obtained in experiments performed at 25 °C and
without detergent in the incubation buffer. As previously noticed (28),
unspecific binding of Syt I to beads is totally abolished in the
presence of nonionic detergent. For this reason, OG was included in the
incubation buffer.
FRET Measurements--
The cytoplasmic domain of Syt I and II
were dialyzed extensively against 20 mM bicine, pH 8.5, 100 mM KCl and then labeled with N-succinimidyl-CY3
and N-succinimidyl-CY5 (Amersham Pharmacia Biotech) for 30 min at room temperature using a protein/dye ratio of 1:17 (58). The
reaction was blocked by the addition of 100 mM glycine and
incubation for 5 min at 4 °C. Excess dye was removed on a PD10 gel
filtration column (Amersham Pharmacia Biotech) pre-equilibrated in 20 mM HEPES-NaOH, pH 7.6, 100 mM KCl, and the
yield of labeling was determined spectrophotometrically by using
extinction coefficients of 31,270 M
1
cm
1 for Syt I and 28,830 M
1
cm
1 for Syt II, both at 280 nm, and extinction
coefficients of 150,000 M
1 cm
1
at 554 nm for CY3 and 250,000 M
1
cm
1 at 650 nm for CY5.
Spectrophotometric experiments were performed by adding increasing
amounts of calcium to a 50-µl cuvette (Hellma, Jena) containing 0.1 µM CY3-Syt II in 20 mM HEPES-NaOH, pH 7.6, 100 mM KCl, 2 mM EGTA, and either 0.5 µM CY5-Syt I or unlabeled Syt I. In selected samples, 0.5 mM free Mg2+ was also added to monitor the
effect of divalent cations different from Ca2+ on the
equilibrium. Fluorescence emission spectra were recorded in a 710 PTI
spectrofluorimeter (Photon Technology International, South Brunswick,
NJ) with an excitation wavelength of 540 nm. Excitation and emission
slit widths were set to 4 nm. The average CY3 fluorescence in the range
of 560-590 nm was then normalized for the maximum fluorescence
emission intensity at 570 nm. This correction allowed the comparison of
different experiments despite variations in the initial CY3-Syt II
concentration. Data were expressed as FRET efficiency
(EF), where EF = 1
RF', and RF'
corresponds to the CY3 fluorescence in the presence or in the absence
of FRET acceptor (RF' = F'CY3-Syt II/CY5-Syt I/F'CY3-Syt II/Syt I).
 |
RESULTS AND DISCUSSION |
Analyzing the roles of individual synaptotagmins is complicated by
the large number of isoforms present in mammals, by their overlapping
distributions, and by the difficulties in obtaining isoform-specific
tools. In the past, isoform-specific antibodies have been extremely
useful for the analysis of the subcellular distribution of several
other synaptic proteins, including amphiphysin, syntaxin, SNAP-25, and
VAMP/synaptobrevin (59-63). For the generation of isoform-specific
antibodies, it is crucial to identify stretches of amino acids with
relatively low homology. The comparison of the 13 synaptotagmin
sequences presently available indicates that the regions of high
similarity are restricted to the two C2 domains and the C terminus,
while the hydrophilic neck region and the intravesicular N-terminal
segment show only limited homology (4, 5). This last segment is
particularly interesting because, as shown for Syt I, it is unlikely
that an antibody directed against it will perturb the physiological
function of the protein (64). Furthermore, antibodies against the N
terminus can be used to monitor cycles of SSV exoendocytosis in neurons
(64-66). We therefore immunized rabbits with peptides corresponding to
the N terminus of Syt I, II, and IV. After affinity purification on the
corresponding immobilized peptide column, synaptic fractions were
probed with the antibodies for specific Syt immunoreactivity. As shown
in Fig. 1A, each antibody
recognized a single major band in both cortical synaptic vesicles and a
membrane fraction from cerebellum, and this reactivity was abolished by
preincubation with an excess of the correspondent generating peptide.
In the case of the anti-Syt IV, the immunoreactivity was reduced
significantly but not completely abolished (not shown). As
complementary proof of specificity, preimmune and immune anti-Syt IV
sera were tested against the same cortical and cerebellar membrane
fractions. A single band with an apparent molecular mass of 62 kDa was
detected only with the immune serum (Fig. 1A).

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Fig. 1.
A, specificity of the isoform-specific
anti-synaptotagmin N-terminal antibodies. Affinity-purified anti-Syt I
and II antibodies were incubated either with dimethyl sulfoxide ( 4%
(v/v) final concentration) ( ) or with the generating peptide
corresponding to the N terminus of the specific isoform (0.5 mg
dissolved in dimethyl sulfoxide) (+) and then used to probe
nitrocellulose membranes containing 15 µg of purified SSV from cortex
(CTX) and 50 µg of a crude vesicle fraction from
cerebellum (CBM). Alternatively, the same brain fractions
were used to test preimmune and immune anti-Syt IV serum. B,
Syt I, II, and IV colocalize in an SSV preparation from rat brain
cortex. The elution profile of a glycerol-coated controlled pore glass
beads column (monitored at 280 nm) loaded with an impure fraction of
rat cortical SSV is shown in the upper panel. Proteins from
the resulting fractions were separated by SDS-PAGE, transferred onto
nitrocellulose, and then analyzed with anti-Syt I, II, and IV-specific
antibodies. The immunoreactivity for Syt I and Syt II co-localizes and
overlaps with the second peak of the elution profile (CPG-II), which
corresponds to pure SSV. Syt IV immunoreactivity also peaks in the
CPG-II area, but it is slightly shifted compared with the signal for
Syt I and II.
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Fractions derived from the glycerol-coated size exclusion
chromatography, the last step of the SSV purification, were analyzed by
SDS-PAGE and probed with the affinity-purified isoform-specific antibodies (Fig. 1B). As expected, the immunoreactivity
corresponding to Syt I was localized with the second large peak of the
chromatographic profile, previously referred to as CPG-II (49), which
corresponds to a homogeneous vesicle population with an average
diameter of 39 ± 3 nm, determined by electron microscopy (not
shown). Syt II and Syt IV are also present in the cortical SSV
preparation (Fig. 1B, lower panel).
The distribution of Syt II exactly overlaps the one observed for Syt I,
while Syt IV immunoreactivity is slightly shifted. The presence of Syt
II in a population of rat cortical SSV is particularly surprising,
based on the relatively low abundance of Syt II at the mRNA level
in the rat brain cortex and its specialized localization in cerebellum
and spinal cord, suggesting a mutually exclusive Syt I and Syt II
distribution (56). The presence of multiple Syt isoforms in a
homogeneous SSV preparation suggests two possible scenarios. The first
implies the existence of SSV containing a single synaptotagmin isoform
and characterized, in terms of calcium sensitivity, by the features of
the unique synaptotagmin present on its surface. The entire neuronal
SSV pool would then consist of distinct synaptotagmin-specific SSV
subpopulations that could be mobilized by different calcium levels.
This possibility is compatible with the recent finding that at least
two populations of SSV can be distinguished biochemically in rat brain
cortex (67). The second possibility envisages the presence of multiple synaptotagmin isoforms on the surface of the same vesicle. In this
case, the SSV calcium dependence would thus be determined by the
repertoire and relative abundance of the different isoforms on its
surface. To discriminate between these two possibilities, SSV
immunoisolation experiments were carried out. This strategy, successfully used in the past to isolate glutamate-containing vesicles
from brain homogenates, was chosen because it does not perturb the
integrity of the SSV by avoiding the presence of detergents (52). In
this condition, the N-terminal directed Syt I antibody presents its
limits due to the inaccessibility of the lumen of the SSV. We therefore
used a well established monoclonal antibody against synaptotagmin,
termed M48 (50), with no appreciable immunoreactivity against Syt II
(not shown). After incubation of the immobilized M48 beads with the
purified CPG-II fraction, beads were analyzed by Western blotting with
anti-Syt II or anti-Syt IV-specific polyclonal antibodies and, as a
control, with antibodies against the v-SNARE VAMP-synaptobrevin, a
protein specifically localized on SSV (61, 68). As shown in Fig.
2, Syt I-specific beads immunopurified
SSV containing the majority of the Syt II immunoreactivity; more than
65% of Syt II immunoreactivity was found in the pellet, as determined
by quantitative Western blotting and scanning analysis. In contrast,
less than half of the Syt IV is associated with the Syt I-positive SSV
(
45%), thus indicating the presence of this isoform in at least two
populations of SSV, one of which lacks Syt I. This immunoisolation
result has two important consequences. First, it confirms the finding
that SSV populations with different biochemical features
(i.e. specific membrane protein composition) can be
identified (67) and, second, that a single SSV is characterized by a
specific panel of synaptotagmin isoforms present on its surface. No
conclusions can be drawn at this point regarding the presence of other
synaptic markers on Syt IV-positive vesicles. Future experiments
characterizing the subcellular distribution of Syt IV are necessary to
conclusively define the vesicular compartment containing this
synaptotagmin isoform.

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Fig. 2.
Immunoisolation of SSV. Highly purified
SSV from rat brain cortex were immunoisolated in the absence of
detergents by using anti-Syt I monoclonal antibody conjugated to
protein G-Sepharose beads (Syt I IgG beads) or mock beads
(empty beads). The resulting immunoisolated SSV fractions
were then analyzed by SDS-PAGE and probed for the presence of Syt II
(anti-Syt II), Syt IV (anti-Syt IV), and
VAMP/synaptobrevin (anti-VAMP 2). Lane
T, total input of purified SSV; P, proteins
associated with the immunoprecipitate; S, supernatant
fraction. Both Syt II and VAMP 2 are specifically enriched in the Syt
I-containing SSV, while Syt IV distributes equally between the pellet
and supernatant, thus indicating the presence of this isoform in a
subpopulation of SSV lacking Syt I.
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Recently, Syt I was demonstrated to homodimerize via pairing of its C2B
domain (46, 47). The presence of multiple synaptotagmin isoforms on the
surface of the synaptic membrane raises the possibility that they could
interact by forming heterocomplexes. To test this hypothesis,
immunoprecipitation experiments were performed on different membrane
preparations (Fig. 3). N-terminal Syt I
antibodies conjugated to protein G-Agarose beads were applied to
membrane extracts obtained by solubilizing purified cortical SSV with
4% OG. After incubation, the immunoprecipitate was extensively washed and then analyzed as described in Fig. 2. The same experiments were
performed in parallel on a crude vesicular fraction from cerebellum, a
tissue enriched in Syt II. In both cases, co-immunoprecipitation between Syt I and II was observed (Fig. 3, upper
panel). Experiments were also performed using immobilized
anti-Syt II antibodies, and the same conclusion holds true (data not
shown), thus suggesting that Syt I and II have an intrinsic ability to
interact. In contrast, Syt IV exhibits very low binding to Syt I in
membrane extracts from both cortex and cerebellum (5-10%; Fig. 3,
lower panel). Even at calcium concentrations
supporting maximal interaction between Syt I and Syt II (>100
µM calcium; see below), no significant calcium-dependent interaction between Syt I and IV can be
detected. Under these conditions, only 5-12% of the total Syt IV is
associated with Syt I (not shown). An interpretation of this result is
that the pairing between different isoforms is characterized by a
certain level of specificity and distinct calcium dependences,
determined by the structure and binding properties of their C2B
domains. This situation would be similar to that of the C2A domain of
synaptotagmins, known to interact differently with phosphatidylserine
and syntaxin in distinct isoforms (19). Interestingly, the C2A domain
of Syt IV does not show any calcium-dependent binding of at
least one of these ligands (69, 70), and it is therefore classified as
a member of the calcium-independent synaptotagmin subfamily (4). The
existence of different classes of C2B domains is suggested by recent
observations that distinct C2B domains have different binding
activities for inositol 1,3,4,5-tetrakisphosphate (23) and
phosphoinositides.2 Future
experiments investigating the heterodimerization abilities of different
isoforms are needed to further clarify this point.

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Fig. 3.
Co-immunoprecipitation of Syt I, Syt II, and
Syt IV. Purified SSV from rat brain cortex (CTX) and a
crude vesicular fraction from rat cerebellum (CBM) were
solubilized with OG and then immunoprecipitated in the absence of
externally added calcium by using immobilized anti-Syt I antibodies.
The resulting pellets (P) and supernatants (S)
were then probed with anti-Syt II (upper panel)
or anti-Syt IV antibodies (lower panel).
Association between Syt I and Syt II is detectable in both membrane
fractions, while Syt I and Syt IV show only a minimal interaction
(5-12% of the total).
|
|
Recently, findings from several laboratories have indicated that the
C2B domain-dependent homodimerization of Syt I is
calcium-dependent (46, 47). To determine if this feature is
shared by the interaction between Syt I and Syt II, solubilized
proteins from purified cortical brain SSV were immunoprecipitated with
immobilized anti-Syt I antibodies in the presence of different calcium
concentrations. Calcium potently promotes the interaction between the
two isoforms and, at equilibrium, more than 45% of Syt II is engaged
in the binding with Syt I, as shown by Western blotting with anti-Syt II antibodies (Fig. 4, upper
panel). The concentration of calcium triggering the half-maximal
effect (EC50) is 6 ± 4 µM, while
maximal association between Syt I and II is reached at a concentration
100 µM Ca2+. Together, these results
suggest that, at calcium concentrations experienced by an SSV during
exocytosis, a large fraction of Syt II may be in a complex with Syt I. This finding, together with the observation that multiple synaptic
isoforms are present on the same SSV, raises the possibility that the
self-association of synaptotagmins with different calcium binding
features might create a variety of calcium sensors characterized by
distinct calcium sensitivities.

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Fig. 4.
Calcium dependence of Syt I and Syt II
co-immunoprecipitation. Purified SSV from rat brain cortex were
solubilized with OG and then immunoprecipitated with immobilized
anti-Syt I antibody in the absence (EGTA) or in the presence
of EGTA-calcium buffers with a concentration of free Ca2+
ranging from 1 µM to 1 mM and then analyzed
by Western blotting with an anti-Syt II-specific antibody
(upper panel). Syt II associated with the Syt I
immunoprecipitate was quantified with the NIH Image software by
comparing the Syt II signal present in the immunoprecipitate with that
obtained from a standard curve (0.25, 0.5, 0.75, and 1 times the
starting material). The fraction of Syt II associated with the pellets
in different conditions was expressed as a percentage of the total Syt
II in the sample. Both the sum of the immunoreactivity present in the
supernatant (S) and in the pellet (P) and the
total input of the sample (T) were used as denominators to
calculate the percentage of synaptotagmin recovery. The two values were
compared, and both were used to determine the EC50 of the
calcium dependence of Syt I/Syt II co-immunoprecipitation. The
variability between the two calculation methods is included in the
error bars in the lower
panel. To compare different experiments (n = 5), data were normalized, and mean values were plotted as a function of
calcium concentration (lower panel). The
EC50 of the equilibrium is 6 ± 4 µM
Ca2+ with maximal Syt I-Syt II association reached at
concentration 100 µM Ca2+.
|
|
The heterodimerization process was confirmed in vitro with
recombinant proteins by using two different approaches. The first involved the co-immunoprecipitation of the cytoplasmic domain of Syt I
(residues 95-421) with the homologous portion of Syt II (residues
103-422). In order to minimize steric hindrance during immunoprecipitation, Syt II was tagged at the amino terminus with a
9-residue version of the hemagglutinin epitope (HA-Syt II). The two
purified isoforms, showing a slightly different mobility in SDS-PAGE
(Fig. 5, inset;
lanes A and B), were mixed, and
Ca2+/EGTA buffers were added to obtain the desired free
calcium concentration. HA-Syt II was then quantitatively recovered with
immobilized HA-specific antibodies, and the pellet was analyzed for Syt
I. As shown in the inset of Fig. 5, the presence of Syt I in
the immunoprecipitate is dependent on the addition of HA-Syt II to the
incubation buffer (compare lanes C and
D). The process is dependent on calcium and shows an
EC50 of 5 ± 3 µM (n = 4), a value very similar to the one observed with the native proteins.
The addition of magnesium to the equilibrium, previously reported to
alter the homodimerization process of Syt I (46), only slightly shifts
the EC50 of the phenomenon toward higher calcium
concentrations (9 ± 5 µM; n = 3),
but it is very efficient in reducing the amount of calcium-independent binding between Syt I and Syt II (Fig. 5). These results demonstrate that the recombinant cytoplasmic domain of Syt I and II are competent for calcium-dependent heterodimerization and can mimic the
behavior of the native proteins, in contrast to a previous report
investigating the dimerization of Syt I, where at least one native
protein was required for efficient and calcium-dependent
dimer formation (47). The reason for this discrepancy is not clear, but
it could be ascribed to differences in the conditions used for
synaptotagmin expression and for immunoprecipitation, together with a
different protein tagging approach.

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Fig. 5.
Co-immunoprecipitation of recombinant Syt I
and II. The recombinant cytoplasmic portions of Syt I and Syt II,
the latter tagged at the N terminus with HA, were mixed, and after
incubation in the presence of different calcium concentrations, HA-Syt
II was immunoprecipitated with HA-specific antibodies. Syt I associated
with the beads was analyzed either by densitometric analysis of the
Coomassie Blue-stained gels or by Western blotting with anti-Syt
I-specific antibodies. Recovery was quantified by comparing the signal
present in the pellets with that obtained from a standard curve of
recombinant Syt I (from 50 to 250 ng for Western blotting and from 0.2 to 1 µg for Coomassie staining). Data were expressed as a percentage
of the maximal Syt I present in the Syt II immunoprecipitate. The
EC50 for the calcium dependence of the equilibrium is
similar in both the absence (closed circles) or
the presence (empty circles) of 0.5 mM free magnesium. The addition of magnesium reduces
significantly the amount of calcium-independent binding between Syt I
and Syt II. Inset, lane A, recombinant
Syt I; lane B, recombinant HA-Syt II;
lane C, Syt I associated with HA-Syt II in the
presence of 100 µM Ca2+ after
immunoprecipitation with anti HA-beads; lane D,
as sample C without the addition of HA-Syt II. Under these
conditions, equal amounts of Syt I and II are recovered in the
immunoprecipitate. Stars indicate the position of the
antibody heavy and light chains, respectively.
|
|
The second strategy used to test the in vitro interaction
between Syt I and II is based on a FRET approach (58, 71). For this
purpose, the recombinant cytoplasmic portions of Syt I and II were
labeled with either the fluorescent dye CY3 or with CY5. For both dyes,
the degree of modification was optimized to obtain an incorporation of
1 mol of dye/mol of protein. Experimentally, we used modified
synaptotagmins with a ratio of dye to protein ranging between 0.5 and
1.2.
To test if Syt I and Syt II interact functionally in solution,
CY3-modified Syt II (fluorescent donor) was mixed with CY5-modified Syt
I (fluorescent acceptor) in the absence of calcium and then in the
presence of increasing calcium concentrations (Fig.
6A). To exclude quenching
effects due to the direct interaction of the added protein with the
donor dye CY3, the same experiment was performed with CY3-Syt II alone
(Fig. 6D) or by adding recombinant Syt I without CY5
modification (Fig. 6C). In both of these samples, the
addition of calcium caused a decrease in the fluorescence emission of
Cy3-Syt II, the extent of which is less pronounced than that seen in
the presence of the Cy5-Syt I acceptor. The emission peak at 664 nm
present in the samples of Fig. 6, A and B, is
partly due to direct excitation of CY5-Syt I (Fig. 6B) and, in the presence of the donor Cy3-Syt II, to FRET. Its decrease (instead
of increase) with increasing calcium concentrations could be attributed
to internal quenching of this emission peak, together with a decrease
of the intensity of the donor spectra, which constitutes its base line.
A calcium-dependent internal quenching is also visible in
the solution of pure CY5-Syt I with increasing calcium (Fig.
6B). The FRET efficiency (EF) of the
dimerization was therefore calculated using the emission peak of the
donor, where an efficient control for the FRET-independent quenching of
the fluorophore is available (Fig. 6C).

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Fig. 6.
FRET measurement on the
heterodimerization of recombinant Syt I and Syt II. Fluorescence
emission following excitation at 540 nm of 0.1 µM CY3-Syt
II alone was monitored from 550 to 750 nm in the absence of calcium
(EGTA; dashed line) and then with the
addition of 0.5 µM CY5-Syt I (A) or unlabeled
Syt I (C) or buffer alone (D) in the presence of
different calcium concentrations (continuous
line; from the top 1 nM (2 mM EGTA), 0.1 mM, 0.49 mM, 0.99 mM, 3.86 mM free calcium). Similarly, the
effects of calcium on CY5-Syt I alone (B) and on CY3-Syt II
alone (D) were investigated (from the top 1 nM (2 mM EGTA), 0.1 mM, 0.49 mM and 0.99 mM free calcium). The average CY3
fluorescence in the range 560-590 nm was then normalized for the
maximum fluorescence emission intensity at 570 nm. Data were expressed
as FRET efficiency (EF) versus
calcium concentration. EF corresponds to 1 RF', and RF'' refers
to the CY3 fluorescence in the presence or in the absence of FRET
acceptor (E).
|
|
The average CY3 fluorescence quenching in the range of 560-590 nm was
then normalized for the maximum emission wavelength (
= 570 nm), and
the data were expressed as the FRET efficiency versus the
calcium concentration. EF was calculated as
EF = 1
RF', where
RF' is the ratio between the fluorescence measured in the presence of FRET acceptor and in its absence (58). As
shown in Fig. 6 (A, C, and E) the
addition of acceptor at calcium concentrations
1 nM
(2 mM EGTA) causes a significant quenching (EF = 0.05) that could be attributed to the
calcium-independent Syt I/Syt II association, a feature already
observed in the immunoprecipitation experiments performed with both the
native and recombinant synaptotagmins (see Figs. 4 and 5). The addition
of calcium induces an increase in the FRET efficiency (Fig.
6E). This parameter is directly linked to the
CY5-dependent quenching of CY3 due to FRET and is corrected for the quenching effect due to the direct interaction of the added
protein with the donor CY3. The EC50 of the calcium
dependence of the Syt I/Syt II interaction corresponds to 140 ± 80 µM (n = 5). Analogous results were
obtained in experiments performed using the opposite labeling strategy
(CY3-modified Syt I and CY5-modified Syt II, not shown). Millimolar
magnesium only slightly influenced the interaction between Syt I and
Syt II (not shown). The reason for the discrepancy between the
EC50 values of the heterodimerization determined by
immunoprecipitation and by FRET is not surprising considering the
differences between the two experimental systems. In fact, in contrast
to the immunoprecipitation experiments, FRET analysis is performed at
equilibrium with both ligands present in a homogeneous phase during the
entire measurement. Future experiments monitoring the real time
heterodimerization of the full-length Syt I and Syt II in living cells
via FRET and life-time measurements will be helpful in addressing this issue.
How could this data on synaptotagmin heterodimerization be integrated
into the general mechanism of docking and fusion of SSV at the active
zones of a nerve terminal? At the present time, no unitary view exists
concerning the molecular details of the tethering, docking, priming,
and fusion of a SSV with the presynaptic membrane and the role(s) of
synaptotagmins in these mechanisms (72). A series of compelling results
indicates that the members of the synaptotagmin family represent the
best candidates for the role of calcium sensors in regulated exocytosis
(3-5). At the molecular level, this function can be correlated with
the calcium-dependent interactions with syntaxin,
phosphatidylserine, and phosphatidylinositol 4,5-diphosphate (11,
13-15, 18-20). In addition, due to the ability of the C2B domain to
bind different ligands at resting calcium concentrations
(i.e. phosphatidylinositol 3,4,5-triphosphate and SNAP-25)
(20, 26-28), synaptotagmins may also be involved in the tethering and
docking of SSV to the active zones of the synaptic membrane. The
formation of homo- and heterodimers of synaptotagmins can be integrated
into this network of interactions as an additional element in the
regulation of the calcium-dependent phase of exocytosis. In
fact, the calcium-dependent association of synaptotagmins
with different calcium binding features could create a variety of
calcium sensors characterized by distinct calcium sensitivities. This
combinatorial hypothesis predicts that the probability of a single SSV
exocytic event is determined by the repertoire of synaptotagmins
present on the SSV surface and the gating properties of the calcium
channels at the synapse. Future experiments will assess the specific
targeting and association of synaptotagmin isoforms characterized by
distinct calcium sensitivities in living cells and monitor the
efficiency and calcium sensitivity of neurotransmitter release under
these conditions. This experimental system will also offer the
possibility to investigate the physiological modulator(s) of several
synaptotagmin isoforms, like Syt IV, VI, VIII, IX, and XI, that appear
not to be modulated by calcium (4) and are likely to be involved in
some aspects of membrane trafficking outside the nervous system.
 |
ACKNOWLEDGEMENTS |
We thank R. Watson for the electron
microscopy analysis of SSV, Drs. G. Stenbeck and T. Iglesias for
discussion and critical reading of the manuscript, and C. Thomas for
excellent technical assistance. We also thank the referees of the
manuscript for excellent suggestions and constructive criticism.
 |
FOOTNOTES |
*
This work was supported in part by a fellowship of the
Spanish Ministry of Culture and Education (to J. H.) and the Imperial Cancer Research Fund.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.: 44-171-2693300;
Fax: 44-171-2693417; E-mail: g.schiavo{at}icrf.icnet.uk.
2
C. Thomas and G. Schiavo, unpublished results.
 |
ABBREVIATIONS |
The abbreviations used are:
SSV, small synaptic vesicle(s);
Syt I, II, and IV, synaptotagmin I, II, and IV,
respectively;
FRET, fluorescence resonance energy transfer;
GST, glutathione S-transferase;
OG, octyl-
-D-glucosopyranoside;
NSF, N-ethylmaleimide-sensitive factor;
SNAP, soluble NSF
attachment protein;
SNARE, SNAP receptor;
VAMP, vesicle-associated
membrane protein;
PAGE, polyacrylamide gel electrophoresis;
HA, hemagglutinin epitope..
 |
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