Molecular Mechanisms in Exocytosis and Endocytosis
Role of C2 domain proteins during synaptic vesicle
exocytosis
Sascha Martens1
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, U.K.
Abstract
Neurotransmitter release is mediated by the fusion of synaptic vesicles with the presynaptic plasma
membrane. Fusion is triggered by a rise in the intracellular calcium concentration and is dependent
on the neuronal SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor)
complex. A plethora of molecules such as members of the MUNC13, MUNC18, complexin and synaptotagmin
families act along with the SNARE complex to enable calcium-regulated synaptic vesicle exocytosis. The
synaptotagmins are localized to synaptic vesicles by an N-terminal transmembrane domain and contain
two cytoplasmic C2 domains. Members of the synaptotagmin family are thought to translate the rise in
intracellular calcium concentration into synaptic vesicle fusion. The C2 domains of synaptotagmin-1 bind
membranes in a calcium-dependent manner and in response induce a high degree of membrane curvature,
which is required for its ability to trigger membrane fusion in vitro and in vivo. Furthermore, members of
the soluble DOC2 (double-C2 domain) protein family have similar properties. Taken together, these results
suggest that C2 domain proteins such as the synaptotagmins and DOC2s promote membrane fusion by the
induction of membrane curvature in the vicinity of the SNARE complex. Given the widespread expression of
C2 domain proteins in secretory cells, it is proposed that promotion of SNARE-dependent membrane fusion
by the induction of membrane curvature is a widespread phenomenon.
Introduction
At the chemical synapse, communication occurs by the regulated release of neurotransmitters from presynaptic neurons
into the synaptic cleft where they can activate receptors on the
postsynaptic plasma membrane. In the presynaptic terminal,
the neurotransmitters are stored in small, 40–50 nm diameter
vesicles. Release of neurotransmitters is achieved by the
fusion of these synaptic vesicles with the plasma membrane
and is triggered by an increase in the calcium concentration
in the presynaptic terminal [1].
The hemifusion model of membrane fusion
The fusion of synaptic vesicles with the plasma membrane
entails the merger of two initially separated membranes.
Membrane fusion processes are ubiquitous, being essential
for many aspects of eukaryotic cell biology. Thus intracellular
membrane traffic, the fusion of mitochondria or the infection
of host cells by enveloped viruses require membrane fusion
[2]. Over the last years, a consensus model about the
mechanism by which membranes fuse has emerged [3]
(Figure 1). According to this model fusion is initiated by
Key words: double-C2 domain (DOC2), exocytosis, membrane fusion, soluble N-ethylmaleimidesensitive fusion protein-attachment protein receptor (SNARE), synaptic vesicle, synaptotagmin.
Abbreviations used: DOC2, double-C2 domain; SNAP-25, 25 kDa synaptosome-associated
protein; SNARE, soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor.
1
Present address: Max F. Perutz Laboratories, University of Vienna, Austria (email
[email protected]).
Biochem. Soc. Trans. (2010) 38, 213–216; doi:10.1042/BST0380213
the local destabilization of the two membranes destined to
fuse. Destabilization favours the transition into a so-called
hemifusion stalk in which the two contacting but not the two
distal monolayers are merged. Hemifusion is followed by the
merger of the two distal monolayers resulting in the opening
of the fusion pore. This is the first time contents of the two
membrane enclosed compartments mix. At the synapse the
neurotransmitters would be in contact with the extracellular
space. After fusion pore opening, the fusion pore may enlarge
at the synapse, leading to full collapse of the synaptic vesicle
into the plasma membrane.
For membrane fusion to occur, several energy barriers
have to be overcome. Somewhat counter intuitively, it has
now been established that the barriers for fusion become
progressively larger during the fusion process. Thus the
formation of the hemifusion intermediate requires less energy
than the opening of the fusion pore, which in turn is
energetically more favourable than fusion pore dilation [4].
During membrane fusion, the main conformational
changes are undergone by the merging membranes, which
are regulated by cellular proteins. For synaptic vesicle
fusion the task for the presynaptic fusion machinery is to
rapidly decrease the energy barriers for fusion only on an
increase in the intracellular calcium concentration. A plethora
of proteins has been identified that are required for the
exocytosis of synaptic vesicles. Among them are members
of the MUNC13, MUNC18, complexin, synaptotagmin and
SNARE (soluble N-ethylmaleimide-sensitive fusion proteinattachment protein receptor) protein families [1]. The main
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Figure 1 The hemifusion model of membrane fusion
Depiction of the fusion of a synaptic vesicle with the presynaptic plasma
membrane, but all cellular fusion events are thought to proceed through
similar intermediates. 1. The first task of the cellular fusion machinery is
to specifically tether the vesicle to the plasma membrane. 2. Secondly,
protein-free patches have to be created on both membranes destined
to fuse in order to allow the membranes to come in close enough
proximity for fusion. 3. Fusion is initiated by the local destabilization
of both membranes. At the synapse the synaptic vesicles are highly
curved and may need less destabilization than the plasma membrane
in order for fusion to occur. 4. Membrane destabilization favours
fusion of the two contacting monolayers but not the distal monolayers.
can mediate membrane fusion in reconstituted systems with
varying efficiency depending on the protein density, the
curvature and lipid composition of the reconstituted vesicles
and other experimental conditions [8–12]. It has, however,
also become increasingly clear that, at physiological protein
densities, SNAREs are rather poor fusogens and that, at
higher densities, SNARE-mediated fusion is accompanied by
extensive lysis [12,13]. Also, mechanistically it is not clear
how the force generated on SNARE complex formation can
be transferred into the membrane as the short linker between
the SNARE domain and the transmembrane domain appears
to be flexible [2,11,14,15].
This intermediate is called the hemifusion stalk. 5. The fusion pore
generated by the merger of the two distal monolayers allows for the first
time the content of the synaptic vesicle to be released. 6. Subsequently,
the fusion pore dilates, ultimately resulting in full collapse of the synaptic
vesicle into the presynaptic plasma membrane.
task is now to assign specific functions to these proteins and
to unravel their precise mechanism of action.
The SNARE complex
Probably the most prominent and best studied of the
exocytic proteins are the SNAREs. The SNAREs required
for synaptic vesicle fusion are synaptobrevin, which is
localized to synaptic vesicles by a C-terminal transmembrane
domain and the two plasma membrane SNAREs syntaxin1
and SNAP-25 (25 kDa synaptosome-associated protein).
Syntaxin1 contains a C-terminal transmembrane domain,
whereas SNAP-25 is targeted to the membrane by four
palmitoylated cysteine residues. The three SNAREs have
the ability to form an extremely stable four-helical bundle
to which synaptobrevin2 and syntaxin1 contribute one helix
each and SNAP-25 two helices [5,6]. This four-helical bundle
is also referred to as the SNARE complex. SNARE complex
formation is thought to bring the plasma and vesicular
membranes into close proximity, aiding membrane fusion [7].
Indeed, SNARE complex formation has been shown to be
essential for synaptic vesicle fusion and for a variety of other
intracellular membrane fusion events [7]. Moreover, SNAREs
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Authors Journal compilation The synaptotagmins
As mentioned above, SNAREs do not act alone during
synaptic vesicle exocytosis (and indeed during any other
SNARE-dependent membrane fusion event investigated so
far). Among the best-studied molecules acting alongside the
SNARE complex are the synaptotagmins and, in particular,
synaptotagmin-1. Synaptotagmin-1 is localized to synaptic
vesicles by an N-terminal transmembrane domain. The main
functional domains of synaptotagmins are two cytoplasmic
C2 domains, which show calcium-dependent SNARE and
membrane binding [16,17]. Synaptotagmin-1 is a member of
a protein family with 17 members in humans. Several
of these, in particular synaptotagmin-2, -3, -5, -7 and -9,
show calcium-dependent membrane and SNARE binding
[2]. Synaptotagmin-1 has gained prominence since it has
been shown to translate the rise in the intracellular calcium
concentration into synaptic vesicle exocytosis. In particular,
studies on synaptotagmin-1-knockout mice have shown that
synaptotagmin-1 is required for the synchronous phase of
neurotransmitter release [18]. Synchronous neurotransmitter
release is tightly coupled with the action potential-triggered
opening of voltage-dependent calcium channels. In contrast,
asynchronous neurotransmitter release, occurring with a
slight delay after calcium channel opening, and spontaneous
release, occurring in the absence of an action potential, are
independent of synaptotagmin-1. The calcium sensors for
asynchronous and spontaneous synaptic vesicle fusions are
unknown.
A number of studies have shown that both the
calcium-dependent membrane-binding and SNARE-binding
activities of synaptotagmin-1 are required to trigger fusion.
Thus mutations in synaptotagmin-1 that reduce the affinity
for membranes also reduce the probability of synaptic
vesicle fusion [19–21]. In contrast, mutations that increase
the affinity of synaptotagmin-1 for membranes result in
an increased probability of synaptic vesicle fusion [22].
On the other hand, synaptotagmin-1 has been shown
to bind the components of the SNARE complex in a
calcium-dependent manner [16,20,23–25] and mutations
that reduce this interaction result in a loss of fusion
triggering by synaptotagmin-1 [20,24,26,27]. In addition,
synaptotagmin-1 shows calcium-independent interactions
Molecular Mechanisms in Exocytosis and Endocytosis
with SNAREs and the plasma membrane phospholipid PIP2
(phosphatidylinositol 4,5-bisphosphate) [28,29].
We have recently shown that, on calcium-dependent
membrane binding and insertion by its C2 domains,
synaptotagmin-1 induces a high degree of membrane curvature [30]. We have further shown that membrane curvature
induction by synaptotagmin-1 is required for the promotion
of SNARE-dependent fusion in vitro [30]. Furthermore,
experiments in PC12 cells have shown that membrane
curvature induction by synaptotagmin-1 is also required for
the calcium-dependent exocytosis of dense core granules
[27]. The PC12 cell experiments further revealed that
synaptotagmin-1-induced membrane curvature promotes
opening of the fusion pore, indicating that synaptotagmin1 has a role during all stages of fusion (Figure 1).
Figure 2 Model of synaptotagmin-1-triggered fusion initiation
During vesicle tethering and priming, the synaptic fusion machinery is
assembled. Shortly before fusion the SNARE complex might be partially
assembled, holding the plasma and vesicular membranes in close proximity. The C2B domain of synaptotagmin-1 may be associated with the
SNARE complex due to its calcium-independent interaction with
the syntaxin1–SNAP-25 heterodimers. On elevation of the intracellular
calcium concentration the C2A and C2B domains of synaptotagmin-1
insert into the presynaptic plasma membrane, resulting in buckling of the
membrane towards the insertion and thus towards the vesicle, reducing
the distance between the two membranes. In addition, the lipids in the
C2 domain-free end cap of the buckle are under curvature stress and
will therefore experience a lower energy barrier for the hemifusion stalk
formation, fusion pore opening and fusion pore dilation.
DOC2 (double-C2 domain) proteins
Evidence has shown that proteins of the DOC2 family
are in many ways analogous to the calcium-dependent
synaptotagmins such as synaptotagmin-1 in terms of
membrane- and SNARE-binding and membrane curvature
induction. DOC2A and DOC2B have, like the synaptotagmins, two C-terminal C2 domains but, unlike the
synaptotagmins, have no N-terminal transmembrane domain
and are thus soluble. Instead they contain an N-terminal
MUNC13-interacting domain. DOC2 proteins have been
shown in many different cellular systems to be calciumdependent pro-exocytic proteins. Thus overexpression of
DOC2B in chromaffin cells increases calcium-dependent
granule exocytosis [31]. Moreover, the overexpression of
DOC2B has a direct effect on the behaviour of the fusion
pore, promoting its expansion [31]. This result indicates
that DOC2B is an integral part of the fusion apparatus.
In addition, DOC2 proteins have been shown to promote
exocytosis in other cell types such as β-cells [32], mast cells
[33] and PC12 cells [34].
Conclusion
Based on these results, the following model for
synaptotagmin-1 and other calcium-dependent C2 domain
proteins such as DOC2B is proposed [2,27,30]. Calcium
binding by the two C2 domains results in the interaction
with and insertion into the plasma membrane [35,36]. It is
possible that C2 domains bind to the vesicular membrane but
given its higher net-negative charge, we consider the plasma
membrane a likelier target for the C2 domains. As a result
of membrane insertion, the C2 domains of synaptotagmin-1
buckle the plasma membrane towards the vesicle (Figure 2).
This has two major effects. First, the distance between the
plasma and vesicular membrane is reduced and,
secondly, the lipids in the protein-free end cap of the buckled
membrane are under curvature stress. This curvature stress
is released during the fusion process. Both the decrease
of the inter-membrane distance and the curvature stress
dramatically increase the probability of fusion. Since
membrane binding by synaptotagmin-1 is calciumdependent, this model provides an explanation for the
calcium-dependent triggering of synaptic vesicle fusion by
synaptotagmin-1. In its simplest form, SNARE complex
binding by synaptotagmin-1 merely serves to target the
membrane curvature to the site where the vesicle is tethered
to the plasma membrane. However, it is also possible
that synaptotagmin-1 binding to the SNAREs directly
affects SNARE complex assembly and/or release of the
complexin fusion clamp [16,37–40]. Thus synaptotagmin-1,
and by analogy other DOC2 proteins, have a central role
during membrane fusion acting as fusogens alongside the
SNARE complex.
Proteins with multiple linked C2 domains are widely
expressed and it is therefore possible that the promotion of
SNARE-dependent membrane fusion by the C2 domaindriven induction of membrane curvature is a widespread
phenomenon.
Acknowledgements
I am grateful to Dr Harvey McMahon and Dr Rohit Mittal for critically
reading the manuscript.
Funding
The European Molecular Biology Organization [long-term fellowship
LTF 21-2006] and the Medical Research Council are gratefully
acknowledged for funding this work.
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Received 10 March 2009
doi:10.1042/BST0380213