(Received for publication, October 16, 1995; and in revised form, November 28, 1995)
From the
Synaptotagmin I (SytI) is a synaptic vesicle protein that binds
Ca and is essential for fast,
Ca
-dependent neurotransmitter release in the
hippocampus, suggesting that it serves as a Ca
sensor
for exocytosis. Although SytI has two cytoplasmic
C
-domains, only the first C
-domain was shown to
exhibit Ca
regulation; it binds phospholipids and
syntaxin in a Ca
-dependent manner. By contrast, the
second C
-domain is inactive in these assays and only binds
putative interacting molecules in a Ca
-independent
manner. We have now discovered in a yeast two-hybrid screen for
SytI-interacting molecules that the C
-domains of SytI
interact with themselves. Using immobilized recombinant
C
-domains from SytI and SytII, we found that only the
second but not the first C
-domains of these synaptotagmins
are capable of affinity-purifying native rat brain SytI and that this
binding is Ca
-dependent, suggesting that only the
second C
-domain is capable of a
Ca
-triggered self-association. A relatively high
Ca
concentration (>100 µM) is
required for binding in the presence of Mg
;
Sr
and Ba
but not Mg
can substitute for Ca
. Our data suggest that
the second C
-domain of SytI is also a
Ca
-regulated domain similar to the first
C
-domain but with distinct binding activities.
Synaptotagmin I (SytI) ()is a member of a family of
neuronal proteins that is characterized by a short N-terminal
intravesicular sequence, a single transmembrane region, and a large
cytoplasmic sequence containing two C
-domains (reviewed in
Südhof(1995)). Nine synaptotagmin isoforms have
been described in mammals (Perin et al., 1990; Geppert et
al., 1991;Wendland et al., 1991; Mizuta et al.,
1994; Hilbush and Morgan, 1994; Li et al., 1995a; Craxton and
Goedert, 1995; Hudson and Birnbaum, 1995), four of which are also found
in non-neural tissues (Li et al., 1995a; Hudson and Birnbaum,
1995). At least three synaptotagmins (SytI, SytII, and SytIII) are
synaptic vesicle proteins, of which SytIII is present throughout the
brain whereas SytI and SytII show restricted complementary expression
patterns with partially overlapping distributions (Ullrich et
al., 1994). Mice in which the SytI gene has been mutated exhibit a
lethal phenotype in which there is a selective loss of fast
Ca
-dependent neurotransmitter release in hippocampal
synapses (Geppert et al., 1994). Spontaneous neurotransmitter
release and neurotransmitter release evoked by
Ca
-independent mechanisms are normal, suggesting an
essential role for SytI only in the Ca
-dependent last
step of membrane fusion. Together with the Ca
binding
properties of SytI (Li et al., 1995a, 1995b), these data
suggest that SytI may serve as an exocytotic Ca
sensor.
Based on the presence of C-domains in SytI
and the observation that the C
-domain confers
Ca
regulation onto protein kinase C, it was
speculated early on that SytI may be a Ca
-binding
protein (Perin et al., 1990). Indeed, experiments with
purified SytI demonstrated that it binds Ca
and
phospholipids (Brose et al., 1992) and that it undergoes a
conformational change as a function of Ca
(Davletov
and Südhof, 1994). Studies on recombinant
C
-domains showed that the first C
-domain of
SytI and of most but not all other synaptotagmins binds phospholipids
as a function of Ca
(Davletov and
Südhof, 1993; Chapman and Jahn, 1994; Ullrich et al., 1994; Li et al., 1995a). In addition,
Ca
triggers binding of syntaxin to the first
C
-domain of synaptotagmins with a Ca
dependence that is distinct from that of phospholipid binding,
implying the presence of two Ca
-binding sites in a
single C
-domain (Li et al., 1995a, 1995b).
Surprisingly, the second C
-domain of all synaptotagmins is
inactive in these assays despite a high degree of sequence homology.
Furthermore, the Ca
-dependent binding properties of
the native cytoplasmic domain of purified brain SytI containing both
C
-domains has the same properties as the recombinant single
first C
-domain (Li et al., 1995a, 1995b). Together
these data suggest that the known Ca
binding
properties of SytI can be entirely accounted for by its first
C
-domain alone and raised the possibility that the second
C
-domain may not represent a Ca
-binding
domain. This possibility was supported by the
Ca
-independent interactions of the second
C
-domains of synaptotagmins with clathrin AP2 (Zhang et
al., 1994; Li et al., 1995a) and with polyanions such as
polyinositol phosphates (Fukuda et al., 1994).
We now
report the results of a yeast two-hybrid interaction screen for
proteins binding to the C-domains of SytI. Unexpectedly,
SytI itself was identified as an interacting partner. In vitro binding assays demonstrated a Ca
-dependent
self-interaction of SytI that is specific for its second
C
-domain. These data suggest that in addition to the first
C
-domain, the second C
-domain of SytI is a
Ca
-regulated domain. However, the two
C
-domains have distinct Ca
-regulated
properties, suggesting a functional diversification of
C
-domains in synaptotagmins.
Figure 1:
Ca-dependent binding
of synaptotagmin I (SytI) from rat brain to recombinant
C
-domains from SytI and SytII. GST-fusion proteins
containing the first C
-domain (C
-A), the second
C
-domain (C
-B), or both C
-domains
(C
-A/B) of SytI or SytII were immobilized on glutathione
beads. Immobilized recombinant proteins were incubated with solubilized
rat brain homogenate containing 3.5 mM Mg
in
the presence or absence of Ca
. Beads were washed 5
times, and bound proteins were analyzed by SDS-PAGE and immunoblotting
with a monoclonal antibody to the N terminus of SytI that does not
recognize the recombinant GST-fusion proteins. Note specific binding
only to constructs containing the second C
-domain of SytI
or SytII. For SytII, a mutant second C
-domain corresponding
to a mutation observed in Drosophila (DiAntonio and Schwarz,
1994) was also analyzed (indicated by asterisks in protein
names). In this mutant tyrosine 312 was changed to asparagine. Numbers on the left indicate positions of molecular
weight markers; arrow points to
SytI.
Figure 2:
Cation
specificity of the binding of SytI to the recombinant second
C-domain of SytI. A recombinant GST-fusion protein with the
second C
-domain of SytI (top panel labeled
GSTSytIC
-B) or recombinant GST alone (bottom panel labeled GST) was immobilized on glutathione beads and incubated
with solubilized brain homogenates containing either 1 mM EGTA, no additions, or 1 mM of the indicated divalent
cations. Beads were washed in the incubation buffers, and bound
proteins were analyzed by SDS-PAGE and immunoblotting. The asterisk identifies the position of full-length SytI; the 40-kDa band
observed in lanes 3 and 6 containing high levels of
bound SytI represents the major proteolytic product of SytI (Perin et al., 1991). Note that addition of Mg
slightly suppresses binding. Numbers on the left indicate positions of molecular weight
markers.
Figure 3:
Ca dependence of SytI
binding to the second C
-domains of SytI and SytII.
Recombinant GST-fusion proteins of the second C
-domains of
SytI and SytII (GSTSytIC
-B and GSTSytIIC
-B,
respectively) were immobilized and incubated with brain homogenates in
Mg
-containing buffers with the Ca
concentrations indicated on top of the panels (E = EGTA). Beads were washed extensively in the
incubation buffers. Bound proteins were analyzed by immunoblotting for
the clathrin assembly protein AP2, SytI, and syntaxin I. Numbers on the left indicate positions of molecular weight
markers.
SytI is a Ca-binding protein of synaptic
vesicles that is essential for fast Ca
-dependent
neurotransmitter release from hippocampal neurons (Brose et
al., 1992; Geppert et al., 1994), suggesting that it
serves as the exocytotic Ca
sensor. Previous studies
demonstrated that the first C
-domain of SytI serves as a
Ca
-dependent phospholipid- and syntaxin-binding
domain (Davletov and Südhof, 1993, 1994; Li et
al., 1995a, 1995b). The second C
-domain is inactive in
these assays but binds AP2 and polyanions in a
Ca
-independent manner (Zhang et al., 1994;
Fukuda et al., 1994). The phospholipid and syntaxin binding
properties of a cytoplasmic fragment from SytI containing both
C
-domains are identical to that of the single recombinant
first C
-domain (Li et al., 1995a, 1995b). Together
these results suggested that SytI may perform its Ca
sensor function primarily via its first C
-domain
whereas the second C
-domain may have a distinct function.
We now demonstrate that the second C
-domain also has a
Ca
-dependent activity suggestive of a
Ca
-binding domain. It mediates the
Ca
-dependent aggregation of SytI and SytII with a
Ca
concentration dependence that mirrors the
Ca
dependence of neurotransmitter release if the
experiments are performed in the presence of physiological
concentrations of Mg
. These data suggest a model of
SytI whereby both C
-domains of SytI can serve as
Ca
binding modules with distinct functions (Fig. 4).
Figure 4:
Domain model of SytI and its binding
activities. SytI binds phospholipids and syntaxin in a
Ca-dependent manner via its first
C
-domain and self-associates in a
Ca
-dependent manner via its second
C
-domain. In addition, the N-terminal domains of
synaptotagmin I self-associate in a Ca
-independent
manner via an undetermined sequence, and the C-terminal domains also
bind AP2, polyanions such as polyinositol phosphates
(IP
), and neurexins in a
Ca
-independent manner (see text for
references).
Even in the absence of Ca, SytI
is not a monomer but a multimer (Perin et al., 1991). The
basic unit of this multimer is an SDS-resistant dimer that can be
detected by SDS-PAGE, and this multimerization is mediated by sequences
N-terminal to the two C
-domains (Brose et al.,
1992) (Fig. 4). Our current demonstration of a
Ca
-dependent binding of SytI to itself via its second
C
-domain raises the possibility that during nerve terminal
depolarization and Ca
influx, SytI multimers may be
cross-linked by Ca
into large superstructures. The
function of such a superstructure in fusion is unknown, but it is
conceivable that it would aid in forming a pore that must occur during
membrane fusion and probably involves assembly of a proteinaceous ring.
A considerable number of interactions has been described for
synaptotagmins, not all of which may be physiologically important.
Considering the point of action of SytI, it seems likely that
Ca-regulated activities are more relevant than
constitutive binding activities. Another criterion that supports the
potential physiological relevance of an interaction is the
colocalization of the binding partners. Based on these two criteria,
the interactions of SytI with phospholipids, syntaxin, and itself
appear to be the most likely to be relevant. The recombinant second
C
-domain of SytI also binds syntaxin (see Fig. 3)
and phospholipids (Damer and Creutz, 1994) in a
Ca
-independent manner. However, when the complete
double C
-domain fragment from native SytI prepared by
partial proteolytic cleavage is analyzed, phospholipid binding and
syntaxin binding are completely dependent on Ca
, and
little Ca
-independent binding is observed (Li et
al., 1995a, 1995b). Two other binding activities were demonstrated
for synaptotagmin I that are Ca
-independent and
conceptually intriguing: binding of neurexins and AP2. Although no in vivo data exist to support the physiological significance
of neurexin binding, AP2 binding may be physiologically significant
since AP2 transiently associates with synaptic vesicles (Pfeffer and
Kelly, 1985; Maycox et al., 1992) and synaptic vesicle
recycling is severely impaired in synaptotagmin mutants in Caenorhabditis elegans (Jorgensen et al., 1995).