Chemistry Reference
In-Depth Information
SNARE complex in an orientation that leaves both the polybasic patch and the
Ca
2
+
-
binding sites free to interact with the membrane [
21
,
22
]. All together, a model is
emerging where synaptotagmin-1 structurally changes both the SNARE complex
and the membranes in a
Ca
2
+
-dependent manner, thereby levering the protein and/or
bending the membrane which induces membrane fusion [
3
,
9
,
11
,
16
,
17
,
21
-
25
].
Importantly, in these models, synaptotagmin-1 acts after SNARE nucleation, i.e. at
state where the membranes are already tethered by a trans SNARE-complex in which
at least part of the cytoplasmic SNARE domains, but not the C-terminal transmem-
brane helices, are already coiled up as depicted in the right panel of the cartoon
sketch of Fig.
3.2
. This is referred to as a fusion-arrested state.
A large number of studies aimed to reconstitute
Ca
2
+
-synaptotagmin-1 trig-
gered membrane fusion in vitro used SNARE-containing artificial membranes.
Synaptotagmin-1 was then added either in the form of soluble cytoplasmic C2-
domains of synaptotagmin-1 (C2AB fragment; residues 97-421) or by inserting full
length membrane anchored synaptotagmin-1. A major limitation in these studies
is that SNAREs alone are sufficient to induce fusion of liposomes [
26
]. Indeed,
membrane fusion is relatively efficient when artificial membranes, containing even
a single copy [
27
] of synaptobrevin-2 together with a combination of syntaxin-1A
and SNAP-25, are allowed to fuse. The addition of the soluble C2AB fragment of
synaptotagmin-1 makes membrane fusion somewhat
Ca
2
+
-sensitive and results in
an increase of fusion efficiency between 100
M and 10mM
Ca
2
+
[
3
,
10
,
11
,
16
,
17
,
23
,
24
,
28
-
32
]. However, in these studies, membrane fusion still proceeds in
the absence of the C2AB fragment and the increase in fusion efficiency upon
Ca
2
+
-
addition was usually less than 10-fold compared to the over 18,000-fold increase in
vivo [
33
]. Recently, it was reported that fusion in the presence of membrane-anchored
synaptotagmin-1 was about 3-5-fold increased at 10
µ
M
Ca
2
+
but decreased again
at higher concentrations of
Ca
2
+
[
30
], presumably because under these conditions
synaptotagmin-1 binds to its own membrane and thus cannot facilitate membrane
docking and fusion by interactions with the plasma membrane [
28
,
30
]. The
Ca
2
+
-
dependency was strongly influenced by the lipid composition of both liposome pop-
ulations: increasing the fraction of anionic lipids in the Q-SNARE or R-SNARE
membranes resulted in higher or lower
Ca
2
+
-sensitivities, respectively. Therefore,
in spite of its significance, the mechanism by which synaptotagmin-1 promotes this
interaction is controversial, and the massive increase in membrane fusion efficiency
of
Ca
2
+
-synaptotagmin-1 has not yet been reproduced in vitro.
However, it is clear that the interactions of synaptotagmin-1 with membranes and
SNAREs are predominantly of an electrostatic, i.e. ionic, nature. Indeed, binding of
synaptotagmin-1 both to anionic lipid membranes [
5
,
7
] and to SNARE molecules
[
21
,
22
,
25
] is heavily influenced by the ionic strength of the solution in which the
proteins, vesicles and membranes are present. Thus, it is surprising that the influence
of ionic interactions on membrane fusion in the presence of synaptotagmin-1 has
not been investigated. All the aforementioned in vitro synaptotagmin-1 membrane
fusion studies were performed in solutions which had a relatively high ionic strength
of typically 100-150mMKCl or NaCl in solution. These ionic strengths are a natural
choice for the experiments since the in vivo physiological conditions of a cell are
µ
