(Received for publication, December 5, 1995; and in revised form, January 18, 1996)
From the
Synaptotagmins I and II are Ca- and
phospholipid-binding proteins of synaptic vesicles that may function as
Ca
receptors for neurotransmitter release via their
first C2 domains. Herein, we describe the phospholipid binding
properties of C2A domains of multiple synaptotagmins (II-VI). We
demonstrate that all synaptotagmins can bind negatively charged
phospholipids (phosphatidylserine (PS) and phosphatidylinositol (PI))
in a Ca
-dependent manner, although it was previously
reported that synaptotagmins IV and VI do not bind phospholipids. The
Ca
-dependent interaction of the C2A domain of
synaptotagmin IV with PS was found to have two components with
EC
values of approximately 5 and 120 µM free
Ca
and exhibited positive cooperativity (Hill
coefficient of approximately 2 for both components). This value is
lower than that of the C2A domain of synaptotagmin II (Hill coefficient
of approximately 3). All other isoforms bound PS with high affinity
(EC
of 0.3-1 µM free
Ca
; Hill coefficient of 3-3.5). In addition,
the C2A domain of synaptotagmin IV cannot bind liposomes consisting of
PS (or PI) and phosphatidylcholine, PC (or phosphatidylethanolamine,
PE) (1:1, w/w), indicating that the binding to negatively charged
phospholipids is inhibited by the presence of PC or PE. In contrast,
other isoforms bound all of the liposomes, which include either PS or
PI, in a Ca
-dependent manner. Mutational analysis
indicated that this phospholipid composition-dependent Ca
binding of synaptotagmin IV results in the substitution of Asp
for Ser at position 244. The cytoplasmic domain of synaptotagmin IV
also shows this unique phospholipid binding. However, it binds PS with
a positive cooperativity and an affinity similar to those of the C2A
domains of other isoforms. Our results suggest that synaptotagmin IV is
also a potential Ca
sensor for neurotransmitter
release.
Synaptotagmins constitute a family of vesicle membrane proteins
that are characterized by a short intravesicular amino terminus, a
single transmembrane region, and a larger cytoplasmic carboxyl terminus
containing two copies of highly conserved repeats homologous to the C2
regulatory region of protein kinase C(1) . At least nine
separate synaptotagmin isoforms have been identified in
rodents(1, 2, 3, 4, 5, 6) and three
(o-p65-A, -B, and -C) in the electric ray(7) . The role of
synaptotagmin I (the best characterized form) in
Ca-regulated exocytosis has been demonstrated in
microinjection experiments utilizing neural cells (8, 9) and by studies of null mutants of Caenorhabditis elegans(10) , Drosophila(11, 12) , and mice(13) .
However, whether all other isoforms can also mediate
Ca
-regulated exocytosis remains unknown.
Recently,
we showed that two C2 domains of synaptotagmin have different functions
in synaptic vesicle trafficking by injecting domain-selective
antibodies against two C2 domains into the squid giant
preterminal(14, 15) , superior cervical ganglion
cells(27) , and chromaffin cells(28) . 1) The C2A
domain functions as a Ca sensor in exocytosis because
anti-C2A IgG, which inhibits Ca
/phospholipid binding
to the C2A domain, blocks synaptic vesicle
fusion(14, 27, 28) . 2) The inositol high
polyphosphate series (inositol 1,3,4,5-tetrakisphosphate, inositol
1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate)
blocks synaptic transmission by binding to the C2B
domain(15, 16, 17, 18, 27, 28) .
The C2B domain is also involved in endocytosis, probably by binding the
clathrin assembly protein, AP2, to the C2B domain because anti-C2B IgG
blocks synaptic vesicle uptake(15, 19, 27) .
Previously, we demonstrated functional differences in the C2B
domains of multiple synaptotagmins in terms of inositol high
polyphosphate binding(20) . We then studied whether or not the
C2A domains of multiple synaptotagmins are diversified. The
Ca/phospholipid binding properties of the C2A domains
of multiple synaptotagmins have been compared by Ullrich et al.(21) and Li et al.(6) , and
synaptotagmins IV, VI, and VIII were described as
Ca
-insensitive isoforms. However, these were
determined under very limited conditions (tested only using liposomes
consisting of phosphatidylserine and phosphatidylcholine (1:2.5, w/w)).
In this study, we examined the phospholipid selectivity of the C2A
domains of synaptotagmins II-VI (three neuronal types and two
non-neuronal types). Although all of the C2A domains can bind acidic
phospholipids such as phosphatidylserine and phosphatidylinositol,
synaptotagmin IV shows unique phospholipid composition-dependent
Ca
/phospholipid binding. We also determined the amino
acid residues that account for this unique phospholipid binding by
mutational analysis. On the basis of these results, we discuss the role
of synaptotagmin IV in Ca
-regulated exocytosis.
Site-directed mutagenesis of
GST-STII-C2A(D231N), (D231S), GST-STIV-C2A(S244D) and deletion of
GST-STII-C2A(180-183) proceeded by means of two-step PCR as
follows(20) . In GST-STII-C2A (D231S) for example, the right
and left halves of the C2A domain were separately amplified with two
pairs of oligonucleotides (primer A, 5`-CGGGATCCGAGCCGGAGAACCTGGGCAA-3`
and mutagenic primer C, 5`-CACTAGTGTCTTGCCTCCTAA-3` (right half);
mutagenic primer D, 5`-CACTAGTGATGGCAATCTATAGCTTTG-3` and primer B,
5`-CGGAATTCTCTCCGCCTTGTAGGTC-3` (left half)). The two resulting PCR
fragments were digested with SpeI (underlined), ligated to
each other, and reamplified with primers A and B. The obtained PCR
fragment encoding the mutant C2A domain of synaptotagmin II (D231S) was
subcloned into the BamHI-EcoRI site of pGEX-2T and
verified by DNA sequencing.
By means of the same two-step PCR that introduced an artificial SpeI site into the C2A domain of synaptotagmins, we produced chimera from synaptotagmins II and III. GST-STII/III-C2A contained amino acids 139-177 of mouse synaptotagmin II followed by amino acids 332-421 of mouse synaptotagmin III. GST-STIII/II-C2A contains amino acids 290-330 of mouse synaptotagmin III followed by amino acids 177-267 of mouse synaptotagmin II.
Figure 1:
Ca-dependent
phospholipid binding to the C2A domains of synaptotagmins II-VI
fused to GST. Liposomes and GST fusion proteins were incubated for 15
min at room temperature in 50 mM HEPES-KOH (pH 7.2) in the
presence of 2 mM EGTA or 1 mM Ca
.
After centrifugation at 12,000
g for 10 min, the
pellets (P; phospholipid binding fraction) and supernatants (S; nonbinding fraction) were separated as described under
``Experimental Procedures.'' Equal proportions of the
supernatants and pellets were resolved by 10% SDS-polyacrylamide gel
electrophoresis. Note that all of the C2A domains from synaptotagmins
can bind PS and PI, which are negatively charged phospholipids, but
essentially not PE and PC. GST-STV-C2A and GST-STVI-C2A weakly
interacted with PE and PC, respectively.
Figure 2:
Ca concentration
dependence of phospholipid binding to the C2A domains of synaptotagmins
II and IV. Binding of PS or PS/PC (1:2.5, w/w) liposomes to
GST-STII-C2A (open and closed circles, respectively)
or to GST-STIV-C2A (open and closed triangles,
respectively) was examined as a function of the free Ca
concentration using Ca
/EGTA buffers.
Phospholipid binding was measured as described in the legend to Fig. 1. The proportion of the phospholipid binding fractions was
quantified using a Bio Image (Millipore). GST-STII-C2A binds both PS
and PS/PC (1:2.5) liposomes with high affinity (EC
of
approximately 0.4 and 1.5 µM free Ca
,
respectively) and with strong positive cooperativity (Hill coefficient
of approximately 3). GST-STIV-C2A also binds PS liposomes alone with
two binding constants (EC
of approximately 5 and 120
µM free Ca
) but cannot bind PS/PC
(1:2.5) liposomes. Note that within a high Ca
concentration range (0.2-1 mM), the phospholipid
binding properties of GST-STII-C2A were suppressed. The data are means
± S.E. of three measurements.
To examine further the effects of the phospholipid
composition, six kinds of liposomes (PS/PE, PS/PC, PS/PI, PI/PE, PI/PC,
PE/PC; 1:1, w/w) were tested. GST-STII, V, VI-C2A bound liposomes
containing negatively charged phospholipids (PS or PI) in a
Ca-dependent manner but did not bind PE/PC liposomes (Fig. 3, left and data not shown). In addition,
GST-STIII-C2A showed Ca
-independent binding to PS/PE
or PI/PE liposomes (combination of negatively charged phospholipids
with PE, Fig. 3, middle). This unique binding may be
due to one-third of the amino terminus of the C2A domain of
synaptotagmin III, a region that is different from those of other
isoforms (Fig. 4). Chimeric analysis between synaptotagmins II
and III confirmed these predictions. GST-STIII/II-C2A, which carries
one-third of the amino terminus of the C2A domain of synaptotagmin III
fused to two-thirds of the carboxyl terminus of the C2A domain of
synaptotagmin II, showed Ca
-independent binding to
PS/PE liposomes, whereas GST-STIII/II-C2A, which is a reverse chimera
of GST-STII/III-C2A, showed Ca
-dependent binding to
PS/PE liposomes (data not shown). However, since the entire cytoplasmic
domain of synaptotagmin III (GST-STIII) lacks this
Ca
-independent binding to PS/PE liposomes (data not
shown), these unique binding properties of GST-STIII-C2A may result
from deletion of the C2A domain from the entire protein. In contrast,
GST-STIV-C2A binds only PS/PI liposomes (Fig. 3, right), indicating that the binding of GST-STIV-C2A to PS or
PI is restricted by the presence of PC or PE.
Figure 3:
Phospholipid composition dependence of
liposome binding to the C2A domains of synaptotagmins II, III, and IV.
GST fusion proteins were incubated in the presence of 2 mM EGTA or 1 mM Ca with liposomes composed
of PS mixed with PE (referred to as PS/PE; 1:1, w/w), PS/PC, PS/PI,
PI/PE, PI/PC, and PE/PC. GST-STII, V, VI-C2A bound liposomes containing
either PS or PI (left and data not shown). GST-STIII-C2A shows
Ca
-independent binding to PS/PE or PI/PE liposomes (middle). GST-STIV-C2A only can bind PS/PI liposomes (right), indicating that the binding of GST-STIV-C2A to PS or
PI is inhibited by the coexistence of PC or PE. Abbreviations are the
same as those for Fig. 1.
Figure 4:
Sequence comparison of the C2A domain of
synaptotagmins I-IV. Alignment of the first C2 domains of murine
synaptotagmins (Syt) I-VI. Residues that are identical
in the four sequences are shaded, and conserved residues in
all sequences are shown by bold letters. Residue numbers are
given on both sides. # and * indicate the essential aspartate residues
for Ca binding to the C2A domain of synaptotagmin I
as shown by crystallographic analysis(23) . Note that the C2A
domain of synaptotagmin IV exhibits an aspartate to serine substitution (arrowhead). The sequence of mouse synaptotagmins I and II are
from (17) ; mouse synaptotagmin III was from (20) ;
mouse synaptotagmin IV was from (4) ; rat synaptotagmin V was
from (5) ; and rat synaptotagmin VI was from (6) .
Figure 5:
Mutational analysis of
Ca/phospholipid-binding domain of synaptotagmins.
Phospholipid binding properties of mutant synaptotagmin in the presence
of 2 mM EGTA or 1 mM Ca
except for
GST-STIV-C2A binding to PS/PC, which was measured at a free
Ca
concentration of 100 µM.
GST-STII-C2A(
180-183) completely lacked the phospholipid
binding capacity, whereas GST-STII-C2A(D231N) and (D231S) showed
composition-dependent phospholipid binding properties similar to those
of GST-STIV-C2A as shown in Fig. 2(open triangles).
GST-STIV-C2A(S244D) bound both PS and PS/PC liposomes with high
affinity (EC
of 0.3-0.6 and 2-4 µM free Ca
, respectively). The EC
values were estimated by plotting the same graphs as shown in Fig. 2. dash, not determined because of a lack of
phospholipid binding activity. Abbreviations are the same as those for Fig. 1.
Figure 6:
Ca-dependent
phospholipid binding to the cytoplasmic domain of synaptotagmin IV. A, GST-STIV, containing the entire cytoplasmic domain of
synaptotagmin IV, shows phospholipid composition-dependent binding
properties similar to those of GST-STIV-C2A as shown in Fig. 3.
Phospholipid binding to GST-STIV was also assayed as described in the
legend to Fig. 1in the presence of 2 mM EGTA or 1
mM Ca
. P, pellet; S,
supernatant. PS+PC indicates that PS and PC
liposomes were mixed well after separate sonication. PS/PC means that PS and PC dissolved in chloroform were mixed before
sonication. Note that GST-STIV can bind PS+PC liposomes
Ca
dependently but not PS/PC liposomes. B,
schematic representation of the limited phospholipid binding of
synaptotagmin IV. Synaptotagmin IV cannot bind the membranes in which
PS and PC are uniformly distributed (a) but can bind those in
which PS is distributed in patches (b). closed
circles, PS or PI; open circles, PC or PE; TM,
transmembrane.
Herein, we have demonstrated that the C2A domains of multiple
synaptotagmins (II-VI) can selectively bind negatively charged
phospholipids (PS and PI). Synaptotagmin IV was found to have the
following unique phospholipid binding capacity. (i) The cytoplasmic
domain of synaptotagmin IV, including both C2A and C2B domains, bound
PS with a positive cooperativity and an affinity similar to those of
the C2A domain of synaptotagmin II. However, when deleted from the
full-length protein, the C2A domain of synaptotagmin IV weakly
interacted with negatively charged phospholipid (EC of
approximately 5 and 120 µM free Ca
) and
lost the high cooperativity. (ii) Synaptotagmin IV showed phospholipid
composition-dependent Ca
/phospholipid binding,
indicating that the Ca
/phospholipid binding
capacities of synaptotagmin IV are restricted by the composition of the
phospholipid in the membrane. A sequence comparison of the C2A domains
of synaptotagmins I-VI (Fig. 4) and mutational analysis (Fig. 5) indicated that these unique binding properties of
synaptotagmin IV are attributable to the Ser residue at position 244,
which is located in the putative second Ca
binding
loop(23) . In addition, the first putative Ca
binding loop of the C2A domain of synaptotagmin IV is one amino
acid longer and has sequences that are different from those of the
other isoforms (between # in Fig. 4). Synaptotagmin VI, which is
reportedly a Ca
-insensitive isoform(6) ,
showed the same Ca
/phospholipid binding properties as
that of synaptotagmin II, at least under our binding conditions. These
results are also supported by the observation that synaptotagmin VI has
4 conserved Asp residues responsible for Ca
binding
(# and * in Fig. 4).
Rather than
Ca/phospholipid binding, considerable research has
been focused on the Ca
-dependent interaction of
synaptotagmin I with syntaxin I(6, 24) , because its
effective concentration (about 200 µM) corresponds to the
Ca
concentration required for neurotransmitter
release (25) . Therefore, it is likely that within a low
Ca
concentration range
(10
-10
M),
synaptotagmin binds phospholipid membranes, whereas in a high
Ca
concentration range
(10
-10
M), it
interacts with syntaxin more efficiently than with phospholipid because
the phospholipid binding capacity is suppressed at a high
Ca
concentration (Fig. 2). At present, we do
not know whether these two Ca
-dependent interactions
are essential for neurotransmitter release. This is the first report,
to our knowledge, showing the unique phospholipid composition-dependent
binding of synaptotagmin IV. Furthermore, although the C2A domain of
synaptotagmin IV fused to GST did not bind syntaxin I(6) , we
believe that its Ca
-dependent interaction with the
syntaxin family remains to be fully elucidated because GST-STIV-C2A
(amino acids 151-281) and GST-STIV (amino acids 39-425)
have markedly different affinities for Ca
in terms of
phospholipid binding.
Synaptotagmin IV is distributed at low
although uniform levels throughout the brain(21) , and little
attention has been focused on this isoform as compared with
synaptotagmin I. However, Vician and co-workers (26) reported
that synaptotagmin IV is an immediate early gene induced by
depolarization in the brain. After kainic acid-induced seizures in
rats, synaptotagmin IV mRNA levels are elevated in the hippocampus and
piriform cortex, whereas synaptotagmin I mRNA is only slightly
decreased. On the basis of these results, together with our own
observations, we propose that synaptotagmin IV is involved in
Ca-dependent presynaptic events.