(Received for publication, October 5, 1994; and in revised form, November 12, 1994)
From the
SNAPs (soluble NSF attachment proteins) are cytoplasmic proteins
that bind to specific membrane receptors and mediate the membrane
binding of NSF (N-ethylmaleimide-sensitive factor), a protein
that is required for membrane fusion reactions. Three synaptic proteins
in brain (SNAP25 (synaptosomal-associated protein of 25 kDa; no
relation to the SNAPs for NSF), synaptobrevin/VAMP, and syntaxin) were
identified as SNAP receptors by affinity chromatography on immobilized
-SNAP complexed to NSF (Söllner, T.,
Whiteheart, S. W., Brunner, M., Erdjument-Bromage, H., Geromanos, S.,
Tempst, P. and Rothman, J. E. (1993) Nature 362,
318-324). However, the nature of the
-SNAP binding site is
unclear. We now show that
-SNAP binds tightly to the complex of
syntaxin with synaptobrevin. SNAP25 is not required for tight binding
of
-SNAP to this complex but stabilizes the syntaxin-synaptobrevin
complex by forming a trimeric core complex with it.
-SNAP does not
bind to synaptobrevin individually and binds only weakly to syntaxin
and SNAP25 in the absence of synaptobrevin. These data suggest that the
complex of the vesicular protein synaptobrevin with the plasma membrane
protein syntaxin is required for physiological
-SNAP binding.
Thus,
-SNAP probably functions in a late step of the membrane
fusion reaction after the formation of the
synaptobrevin-syntaxin-SNAP25 core complex.
In pioneering studies on vesicular membrane transport between
Golgi stacks in vitro, Rothman's laboratory demonstrated
that a soluble ATPase called NSF ()is essential for membrane
transport (Wilson et al., 1989). Further studies revealed that
NSF acts by binding to the interacting membranes in a reaction mediated
by a second class of cytoplasmic proteins called SNAPs (Clary et
al., 1990). SNAPs in turn bind to membranes via specific receptors
that are thought to be present on vesicular and target membranes and
are referred to as v- and t-SNAP receptors, respectively
(``SNAREs''). In vitro, NSF and SNAPs are also
essential for early endosome fusion (Diaz et al., 1989) and
for membrane traffic between the endoplasmic reticulum and the Golgi
complex (Beckers et al., 1989), suggesting that these proteins
perform a universal function in membrane traffic. Furthermore,
mutations in the yeast homologs for NSF and SNAPs result in a secretory
defect, demonstrating that these proteins are functionally conserved in
evolution (reviewed by Pryer et al.(1992)). Although NSF and
SNAPs are required for membrane traffic, their mechanisms of action are
unclear.
In the nerve terminal, synaptic vesicles execute a complex
life cycle that involves docking, membrane fusion, and budding steps at
the plasma membrane and at the endosome. Proteins that participate in
these steps are now being characterized. Particular progress has been
made in the elucidation of the proteins functioning in the docking and
fusion of synaptic vesicles at the plasma membrane. Two independent
approaches identified the same three proteins, synaptobrevin/VAMP,
syntaxin, and SNAP25 (synaptosomal-associated protein of 25 kDa; no
relation to the SNAPs for NSF), as likely core components of the
membrane fusion machinery (reviewed by Südhof et al.(1993), Bennett and Scheller(1994), and Ferro-Novick and
Jahn(1994)). First, studies on clostridial neurotoxins that block
synaptic vesicle membrane fusion revealed that these constitute a
family of metalloproteases which specifically cleave either
synaptobrevin, syntaxin, or SNAP25 (Link et al., 1992; Schiavo et al., 1992; Blasi et al., 1993a, 1993b). Second,
affinity chromatography on immobilized -SNAP complexed to NSF
resulted in the purification of the same three proteins, indicating
that syntaxin, synaptobrevin, and SNAP25 are SNAP receptors
(Söllner et al., 1993a). These studies
suggest a central role for syntaxin, synaptobrevin, and SNAP25 in
synaptic vesicle exocytosis.
Syntaxin and SNAP25 are plasma membrane proteins that are present as a tight complex (Hayashi et al., 1994; Pevsner et al., 1994). Synaptobrevin is a synaptic vesicle protein that binds only weakly to syntaxin or SNAP25 separately but very tightly to the complex of syntaxin and SNAP25, resulting in the formation of a stable SDS-resistant complex (Hayashi et al., 1994). These findings have led to the concept of a core complex consisting of at least three components: synaptobrevin, syntaxin, and SNAP25. The core complex bridges the synaptic vesicle and plasma membranes and may function in the docking and/or fusion of synaptic vesicles.
The stage at which -SNAP and NSF bind to
components of the core complex is not known. It is possible that
-SNAP binds to one or more of the components separately, implying
a function for SNAPs and NSF in the docking of synaptic vesicles with
the plasma membrane. Alternatively,
-SNAP and NSF may not bind
until the core complex has formed. The later result would imply that
the function of
-SNAP and NSF is exerted only after docking. In
the current experiments, we show that
-SNAP binds only weakly to
isolated syntaxin or SNAP25 or the syntaxin-SNAP25 complex, and not at
all to synaptobrevin. Binding is markedly enhanced after synaptobrevin
complexes with syntaxin or the syntaxin-SNAP25 complex. These results
imply a post-docking role for
-SNAP and NSF.
Figure 1:
Weak binding of -SNAP to syntaxin
and SNAP25 but not to synaptobrevin. GST-
-SNAP, GST-SNAP25, and
GSTsyntaxin fusion proteins (0.1 nmol) encoded by the indicated pGEX
vectors were bound to glutathione-agarose and incubated with extracts
from COS cells transfected with syntaxin IA, SNAP25, and synaptobrevin
II expression vectors (pCMV-syntaxin, -SNAP25, and -Syb2, respectively). After washing, proteins bound to the
beads were analyzed by SDS-PAGE and immunoblotting with the indicated
antibodies. The binding of SNAP25 and syntaxin to each other is
stronger than either to
-SNAP, while binding of
-SNAP to
synaptobrevin was not detected.
Figure 2:
Binding of -SNAP to different
isoforms of syntaxin, SNAP25, and cellubrevin. GST fusion proteins of
syntaxins IA-IV (GST-SyntIA, -II, -III, and -IV), SNAP25 (GST-SNAP25), and
cellubrevin (GST-Ceb) were bound to
glutathione-agarose beads and incubated with purified
-SNAP. Beads
were washed, and bound proteins were analyzed by SDS-PAGE followed by
Coomassie Blue staining (top and middlepanels) and immunoblotting with antibodies to
-SNAP (bottompanel). The top and middlepanels depict Coomassie-stained gels of the GST fusion
proteins before and after the binding experiments, respectively.
Purified
-SNAP in amounts corresponding to the quantity added to
each experimental condition was loaded in the rightlanes of both gels for comparison. The bottompanel displays an immunoblot for
-SNAP of the material shown in the middlepanel. Arrows point to
-SNAP,
and numbers on the left show positions of molecular
weight standards.
Figure 3:
Binding of -SNAP to syntaxin IA. GST
fusion proteins encoding the complete cytoplasmic domains of syntaxin
IA (GST-Synt1A, amino acids 1-265), a mutant syntaxin
containing a deletion of most of the N-terminal sequences (GST-Synt
A, amino acids 1-8 and 76-265) and a
mutant syntaxin with a deletion of the C-terminal sequences (GST-Synt
B, amino acids 1-180) were bound to
glutathione-agarose beads and incubated with purified
-SNAP (rightlane). Beads were washed and the pellet
fractions were analyzed by SDS-PAGE, followed by Coomassie Blue
staining (toppanel) or immunoblotting for
-SNAP (bottompanel). Arrows point to the position
of
-SNAP.
Figure 4:
-SNAP-binding to syntaxin:
potentiation by synaptobrevin. Extracts from COS cells transfected with
cytomegalovirus expression vectors encoding
-SNAP, SNAP25, and
synaptobrevin 2 (Syb2) (containing approximately 10
pmol of expressed protein) were incubated with 100 pmol of GST-syntaxin
overnight. Bound proteins were analyzed by immunoblotting with
antibodies to
-SNAP (toppanel), SNAP25 (middlepanel), and synaptobrevin 2 (bottompanel) using enhanced chemiluminescence detection. Note
that the exposure shown was chosen to demonstrate the potentiation of
-SNAP binding to syntaxin by synaptobrevin. Almost no
-SNAP
binding to syntaxin in the absence of synaptobrevin is apparent in this
exposure; it can, however, be detected with longer exposures (data not
shown).
Figure 5:
Quantitative analysis of -SNAP
binding to syntaxin in the presence and absence of synaptobrevin.
His-tagged
-SNAP at the indicated concentrations was bound to
immobilized GST-syntaxin (300 pmol) in the presence and absence of
synaptobrevin 2 (approximately 20 pmol) in a 0.25-ml reaction volume.
Samples were analyzed by quantitative immunoblotting using
I-labeled secondary antibodies and detected with a Fuji
Bio-Imaging Analyzer. Note that under the conditions used in this
experiment, it was not possible to saturate
-SNAP binding to
syntaxin IA in the absence of
synaptobrevin.
As a positive control
for the binding reaction, the binding of syntaxin 1A, SNAP25, and
synaptobrevin 2 to each other was analyzed (Fig. 1). In
agreement with our previous experiments (Hayashi et al.,
1994), syntaxin and SNAP25 were found to bind strongly to each other
independent of which protein was expressed as a native protein in COS
cells or as a GST fusion protein, thereby validating the binding
reactions. Furthermore, synaptobrevin binding to syntaxin was almost
undetectable in the absence of SNAP25 but greatly potentiated by
addition of SNAP25, leading to the formation of the stable
synaptobrevin-syntaxin-SNAP25 core complex. In contrast, the addition
of SNAP25 had no potentiating effect on synaptobrevin binding to
-SNAP (Fig. 1, right lanes).
In a complementary
set of experiments, we investigated the binding of purified His-tagged
-SNAP to GST fusion proteins with different syntaxins, SNAP25, and
cellubrevin (Fig. 2). Cellubrevin is a ubiquitous homolog of
synaptobrevin that can replace synaptobrevin in the
syntaxin-SNAP25-synaptobrevin complex (McMahon et al., 1993)
and was used instead of synaptobrevin because its GST fusion protein is
more stable than that of synaptobrevin. Coomassie Blue staining and
immunoblotting revealed that most syntaxin isoforms and SNAP25 but not
cellubrevin were capable of binding
-SNAP. More robust binding
signals were observed in these experiments than in those described in Fig. 1because high, saturating concentrations of
-SNAP were
used (Fig. 2, right lane). This experiment confirms the
conclusion of Fig. 1that syntaxin and SNAP25 but not
synaptobrevins can serve as SNAP-binding proteins.
A large number of
proteins has been shown to bind to syntaxin, including the members of
the core complex (synaptobrevin and SNAP25), synaptotagmin, Munc18, and
potentially Ca channels. Thus it is important to map
the sites of interaction of these proteins on syntaxin, and preliminary
studies have indicated that the core complex components interact
primarily via the C-terminal sequences of syntaxin, whereas the N
terminus of syntaxin is required for Munc18 binding (Hata et
al., 1993; Hayashi et al., 1994). We therefore tested the
ability of N- and C-terminal deletion mutants to bind
-SNAP,
revealing that the C terminus of syntaxin is essential for
-SNAP
binding, whereas the N terminus is not as critical (Fig. 3).
These data suggest that the
-SNAP-binding site on syntaxin is
similar to that for SNAP25 and synaptobrevin (Hayashi et al.,
1994).
To quantitate the
difference in -SNAP binding to syntaxin in the absence or presence
of synaptobrevin, we performed binding experiments that were analyzed
with
I-labeled secondary antibodies. Increasing amounts
of recombinant His-tagged
-SNAP were added in these experiments to
a constant amount of GST-syntaxin bound to glutathione-agarose beads in
the presence or absence of synaptobrevin 2. After incubations, bound
proteins were recovered from the washed beads and quantitatively
analyzed by immunoblotting (Fig. 5). Although syntaxin binds
-SNAP in the absence of synaptobrevin, the concentrations of
-SNAP required for binding are much higher than in the presence of
synaptobrevin. Binding of
-SNAP to syntaxin saturates at low
-SNAP concentrations (below micromolar) in the presence of
synaptobrevin but does not saturate in the absence of synaptobrevin
under the conditions of the experiments. Thus, synaptobrevin binding to
syntaxin greatly increases
-SNAP binding, either by forming a
composite receptor surface for
-SNAP or by inducing a
conformational change in syntaxin that results in high affinity
binding.
A difference in affinity for -SNAP between syntaxin
and the core complex of syntaxin-synaptobrevin-SNAP25 would only be
physiologically relevant if
-SNAP were a limiting component in
brain and not present in saturating concentrations. To address this
question, we performed quantitative immunoblotting experiments on
syntaxin IA and
-SNAP in total brain homogenate using GST-syntaxin
and His-tagged
-SNAP as standards for controlling for antibody
affinities. Although these experiments did not allow the calculation of
a true concentration of these proteins in brain, they did reveal that
syntaxin IA is at least 100 times more abundant than
-SNAP (data
not shown). In spite of its low abundance,
-SNAP was found to be
highly enriched in the putative synaptic vesicle membrane fusion
complex isolated by immunoprecipitation with syntaxin antibodies from
brain, with an enrichment that exceeded that of the other components of
the complex (synaptobrevin and SNAP25). Thus the affinity for
-SNAP is likely to be a governing determinant in the definition of
the
-SNAP receptor.
There is general agreement that the synaptobrevin, syntaxin,
and SNAP25 form a synaptic core complex that is important for synaptic
vesicle exocytosis. However, the role of the complex in synaptic
vesicle docking (e.g. Pevsner et al.(1994)) or
membrane fusion (e.g. Hayashi et al.(1994)) is
unclear. Furthermore, the function of the interaction of synaptotagmin
I, -SNAP, and Munc18-1 with components of the complex has been
controversial (Hata et al., 1993; Pevsner et al.,
1994; Söllner et al., 1993b), although
recent studies on murine synaptotagmin I mutants reveal that
synaptotagmin I has an essential function in the late stages of the
fusion reaction (Geppert et al., 1994).
In the current
study, we confirm previous results (Hayashi et al., 1994)
demonstrating that synaptobrevin 2 binds much more tightly to the
complex of syntaxin IA and SNAP25 than to either component individually
and forms an SDS-resistant stable complex, referred to as core complex.
We then show that -SNAP binds only weakly to either syntaxin IA or
SNAP25 alone and not to synaptobrevin 2. However, addition of
synaptobrevin 2 to syntaxin IA or to the physiologically occurring
syntaxin-SNAP25 complex causes a dramatic enhancement in
-SNAP
binding, probably due to a shift in affinity. Quantitative
immunoblotting revealed that
-SNAP is present at a concentration
more than 100-fold lower in brain than in syntaxin, suggesting that
-SNAP is limiting. Together these results demonstrate that the
synaptic core complex of synaptobrevin, syntaxin, and SNAP25 and not
the individual components serves as an
-SNAP receptor. The high
affinity SNAP binding site could be formed by a composite receptor
surface on syntaxin and synaptobrevin or could be induced in syntaxin
allosterically by synaptobrevin binding.
A model based on these
findings is depicted in Fig. 6. Syntaxin is thought to be
normally present as a heterodimeric complex with either Munc18 or
SNAP25 in the plasma membrane. During or after synaptic vesicle
docking, the syntaxin-SNAP25 dimer forms a stable trimeric complex with
synaptobrevin. This complex, the core complex, becomes SDS-resistant
through a conformational change (Hayashi et al., 1994) and
serves as the binding site for the consecutive binding of SNAPs and
NSF. It is unclear if the formation of the SDS-resistant complex
precedes SNAP binding since -SNAP also binds to the less stable
synaptobrevin-syntaxin complex that is not SDS-resistant. However,
since after complex formation SDS resistance occurs spontaneously, it
seems likely that the SDSresistant complex represents the true SNAP
receptor. The sequential binding of first SNAP and then NSF to the
complex is suggested by the absence of a SNAP-NSF complex in the
cytosol. NSF is a homotrimer in which all three subunits have to be
functional for NSF to be active in fusion reactions (Whiteheart et
al., 1994). At this point it is unknown if the core complex has
one or several binding sites for
-SNAP, since SNAP25 exhibited
weak binding of
-SNAP, which could not be significantly enhanced
by synaptobrevin (Fig. 1) but could still be utilized after
formation of the high affinity complex between syntaxin-synaptobrevin
and SNAP25. The preferential binding of
-SNAP to the core complex
and not to individual components implies a role for SNAPs and NSF late
in the fusion reaction, most likely in the step that prepares the
vesicle and plasma membranes for the actual fusion reaction. It is
tempting to speculate that the function of NSF is to prepare the
membranes bridged by the core complex for the actual fusion reaction,
possibly by multimerizing several core complexes into a prefusion
state.
Figure 6: Model of the assembly sequence of the putative core complex for synaptic vesicle membrane fusion. In the first step, synaptobrevin (Syb) on the vesicle membrane (VM) and syntaxin 1 and SNAP25 on the plasma membrane (PM) are thought to form a complex (7SComplex). This step may be identical with the docking of synaptic vesicles or may immediately follow docking of synaptic vesicles. The synaptobrevin-syntaxin-SNAP25 complex is then converted into a stable, SDS-resistant form (Hayashi et al., 1994), which serves as a receptor for the sequential binding of SNAPs and of NSF (steps 3 and 4).