 |
INTRODUCTION |
SNAREs1 represent a
protein superfamily that is thought to play a key role in all
intracellular membrane fusion events within eukaryotes (1-6). They
possess a homologous domain of approximately 60 amino acids referred to
as the SNARE motif (7). The best characterized SNAREs are those
mediating exocytosis of synaptic vesicles in neurons. They include the
vesicle protein synaptobrevin (also referred to as VAMP) and the
membrane proteins SNAP-25 and syntaxin 1. In vitro, these
proteins form a stable ternary complex that is reversibly dissociated
by the soluble ATPase NSF in conjunction with soluble cofactors termed
SNAPs (8, 9). Assembly and disassembly of SNAREs has recently been
investigated in detail by several laboratories (5, 6, 10-12). It is
generally believed that it is the formation of a ternary complex
between complementary SNAREs residing on the membranes destined to fuse
("trans" complexes) that drives the fusion reaction. After fusion,
the complexes are disassembled by NSF and SNAPs and thus re-energized
for another round of membrane fusion.
According to the original SNARE hypothesis (1), each fusion step in
membrane trafficking would be mediated by a unique set of SNAREs. These
would function only in one fusion step and be excluded from others.
This specificity was thought to be caused by the intrinsic affinity of
SNAREs for each other, i.e. only cognate SNAREs were thought
to bind to each other. Recently, however, it has become clear that at
least some SNAREs can function in multiple trafficking steps such as
the yeast proteins Sed5p and Vti1p (13-15). Furthermore, these
proteins apparently participate in the formation of several different
SNARE complexes, suggesting that they are able to pair with more than
one set of partners.
SNARE complex assembly is mediated by the SNARE motifs of the
participating proteins which form a protease-resistant core domain (16,
17). The transmembrane regions of syntaxin and synaptobrevin are
directly adjacent to the SNARE motifs, aligned at one end of the core
domain (18, 19). A dramatic increase in
-helical content is
associated with SNARE complex formation, showing that major
conformational changes occur during assembly. (12, 20-23). These
features, together with the heat stability of the complex (21), led to
the proposal that the SNAREs "zipper up" during assembly, forcing
the transmembrane domains into close proximity and thus pull the fusing
membranes together (3, 4). The energy released during assembly would
thus be used to overcome the energy barrier separating the two
membranes (21).
The central domain of the synaptic SNARE complex is represented by a
12-nm-long bundle consisting of four parallel
-helices that are
wound around each other (24). The interacting amino acids form distinct
layers perpendicular to the axis of the four helix bundle, which are
similar to those found in typical coiled-coils. These layers are formed
by hydrophobic amino acid side chains with the exception of an ionic
layer in the middle which consists of three glutamine residues,
contributed by syntaxin and the two SNARE-motifs of SNAP-25, and one
arginine residue, contributed by synaptobrevin (24). The striking
conservation of the glutamine (Q) and arginine (R) throughout the
entire SNARE superfamily led us to reclassify SNAREs into Q- and
R-SNAREs (25). The hydrophobic layers in the four helix bundle are also
conserved whereas residues exposed at the surface are much more
variable. The ability to form four helix bundles is probably the
essential feature of the SNAREs that is conserved among the entire
superfamily (25).
Given the high degree of core residue conservation the question arises
if SNARE pairing is as specific as previously assumed. Numerous
side-chain interactions were observed at the surface of the synaptic
SNARE complex, particularly between SNAP-25 and syntaxin (24), which
involve non-conserved residues (25). It is not known to which extent
these interactions contribute to the overall stability of the SNARE
complex or to the kinetics of SNARE assembly. Since the overall
sequence homology between more distant members of the SNARE superfamily
is relatively low, such interactions may contribute to pairing
specificity. If, however, the surface interactions of the SNARE complex
do not exert a decisive influence on complex assembly and stability,
one would predict that complexes form not only between cognate but also
between non-cognate SNAREs. If complex formation is indeed
indiscriminatory, then one must look elsewhere for explaining the
specificity of membrane fusion events.
In order to address this issue, we have investigated whether different
SNAREs can form complexes, and if so, whether these complexes resemble
the neuronal SNARE complex. In our experiments, we have focused on
complexes containing either closely related or distant relatives of the
neuronal SNAREs. Our results show that all SNAREs investigated here can
be combined in arbitrary composition to yield complexes of very similar
biophysical properties. All complexes have a high
-helical content,
contain a protease-resistant core domain, are heat-stable, and are
disassembled by NSF. Furthermore, the distant synaptobrevin relative,
endobrevin/VAMP 8, is not discriminated from synaptobrevin upon
assembly with the neuronal SNAREs SNAP-25 and syntaxin 1.
 |
EXPERIMENTAL PROCEDURES |
Materials--
NSF and
-SNAP in pQE-9 plasmids encoding for
His6-tagged fusion proteins were kindly provided by S. Whiteheart and J. E. Rothman (Memorial Sloan-Kettering Cancer
Center, New York). Syntaxin 1A (residues 1-265) in the pET22b vector
encoding for a factor Xa cleavable COOH-terminal His6
fusion protein was kindly provided by A. T. Brünger (Yale
University, New Haven, CT). The recombinant protein fragments were
derived from cDNAs encoding for rat synaptobrevin 2 and rat
syntaxin 1A (kindly provided by R. H. Scheller, Stanford University School of Medicine, Stanford, CA) and for SNAP-25A (kindly
provided by T. C. Südhof, University of Texas Southwestern Medical Center, Dallas, TX). Recombinant light chain of botulinum neurotoxin E (BoNT/E) was a generous gift of H. Niemann (Institut für Biochemie, Medizinische Hochschule Hannover, Hannover, Germany).
Molecular Cloning of cDNA Encoding for Rat
Endobrevin--
Rat endobrevin was amplified by PCR using primers
annealing outside of the coding region based on sequence information
from Expressed Sequence Tag data base. cDNA from rat liver, lung,
and kidney was used as a template. The PCR product was subcloned into pBS vector and sequenced. All constructs derived from the three different tissues were identical. The rat endobrevin amino acid sequence was 99% identical to mouse endobrevin (26, 27).
Immunoprecipitation--
PC12 cell homogenates were prepared by
passing the cell suspension 10 times through a ball cracker.
Postnuclear supernatant was generated by centrifugation at 1000 × g for 10 min and solubilized in extraction buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1 mM PMSF, 1% (v/v) Triton X-100)
at a final protein concentration of 0.5 mg/ml. Lysates were clarified
by centrifugation at 200,000 × g for 60 min. After
transfer of the supernatant to a fresh tube, immunoprecipitations were
conducted for 2 h at 4 °C with a monoclonal antibody against
synaptobrevin (69.1) (28) or a serum against endobrevin (residues
1-74) that was raised in rabbits. The serum against endobrevin had
been affinity-purified. Antibodies were bound to Protein G-Sepharose
beads (Amersham Pharmacia Biotech) for 30 min, sedimented, and washed
eight times with extraction buffer. The supernatants were precipitated
according to Wessel and Flügge (29). The immunoprecipitates and
10% of the precipitated supernatants were analyzed by SDS-PAGE and
immunoblotting using the antibody described above for endobrevin, 69.1 for synaptobrevin, and the monoclonal antibody HPC-1 for syntaxin-1
(30).
Generation of Recombinant Fusion Proteins--
Coding sequences
were amplified by PCR using primers with appropriate restriction sites
for subsequent subcloning into the desired plasmid. The sequence
encoding for the cytoplasmic region of rat endobrevin (residues 1-74)
was subcloned into pGEX-KG (Amersham Pharmacia Biotech) via
BamHI and EcoRI restriction sites resulting in a
fusion protein with glutathione S-transferase (GST). The cytoplasmic domains of rat syntaxin 2 (1-265), syntaxin 3 (1-260), and syntaxin 4 (1-273) were subcloned into the pHO2c vector (21) via
NdeI and EcoRI restriction sites resulting in
fusion proteins carrying a carboxyl-terminal His6 tag. In
order to obtain better expression, rat SNAP-25A was subcloned into the
vector pET28a (Novagen) via NheI and XhoI
restriction sites resulting in an amino-terminal His6 tag.
In addition, four cysteines (Cys-84, -85, -90, and -92) were replaced
by serines by the overlapping primer method of Higuchi (31). No
difference in structural and binding properties to the cysteine
containing SNAP-25 construct (21) was observed (data not shown).
Protein Purification--
GST-endobrevin (residues 1-74) was
purified by affinity chromatography on glutathione-Sepharose beads
essentially as described (20). After purification, the GST tag was
cleaved by thrombin. Fusion proteins containing His6 tags
(syntaxin 1 (1-265), pET22b; syntaxin 1 (180-262), pET28a (24);
syntaxin 2 (1-265), pHO2c; syntaxin 3 (1-260), pHO2c; syntaxin 4 (1-273), pHO2c, SNAP-25A, pET28a; synaptobrevin 2 (1-96), pET28a
(24)) were purified by Ni2+-Sepharose as described (16,
21). After elution from the affinity matrices, all recombinant proteins
were dialyzed against standard buffer and further purified by ion
exchange chromatography using Mono-Q or Mono-S columns on an FPLC
system (Amersham Pharmacia Biotech). After loading, the proteins were
eluted with a linear gradient of NaCl in 20 mM Tris, pH
7.4, 1 mM EDTA, 1 mM dithiothreitol (standard
buffer). The peak fractions were pooled and dialyzed against standard
buffer containing 100 mM NaCl. The eluted proteins were
about 95% pure, as determined by SDS gel electrophoresis. All binary
and ternary complexes were purified using a Mono-Q column (Amersham
Pharmacia Biotech) after overnight assembly of the purified monomers.
Protein concentrations were determined by absorption at 280 nm and the
Bradford assay (32).
Limited Proteolysis--
The purified ternary complexes were
subjected to limited digestion in standard buffer containing 100 mM NaCl using proteinase K in a ratio of 1:100 (w:w)
protease:protein complex at 25 °C for 5 min. For analysis by
SDS-PAGE, PMSF-containing SDS sample buffer was added. For analysis by
size-exclusion chromatography on a HR-10/30 Superdex-200 column
(Amersham Pharmacia Biotech) followed by multi-angle laser light
scattering (MALLS), the reaction was stopped by adding 1 mM
PMSF and placing the samples on ice.
Disassembly Reaction--
Ternary complexes were disassembled by
addition of 3 µM NSF, 11 µM
-SNAP, 2 mM MgCl2, 2.5 mM ATP in standard
buffer for 40 min at 30 °C in the presence of 1.5 µM
BoNT/E light chain. The reaction was stopped by heating the samples for
5 min at 95 °C in SDS-sample buffer. As controls, the reaction was
carried out either in absence of NSF and
-SNAP, or the ATPase
activity of NSF was abolished by replacing MgCl2 with 10 mM EDTA. As an assay for disassembly, cleavage of SNAP-25
by BoNT/E was monitored using Tricine electrophoresis for fragment
separation. For immunodetection of SNAP-25, the monoclonal antibody Cl
71.1 (33) was used.
CD Spectroscopy--
Far UV CD spectra were obtained by
averaging over 5-50 scans using steps of 0.2 nm with a scan rate of 50 nm/min on a Jasco model J-720 upgraded to a J-715U equipped with a
6-Position Peltier Effect Cell Changer. Measurements were performed in
Hellma quartz cuvettes with path lengths of 0.1 cm. All CD spectra were
recorded after reaching equilibrium following an overnight incubation
at 4 °C in the standard buffer. To evaluate changes of the CD
spectrum attributable to complex formation, the spectra were compared
with the theoretically noninteracting sum of the individual spectra using the equation [
]sum =
i
cini
[
]i/
i cini, where
ci are the respective concentrations of the
proteins, ni are the respective numbers of amino
acid residues, and [
]i are the mean residue
ellipticities of the individual proteins. For thermal melts, the
ellipticity at 220 nm was measured between 25 and 95 °C with a
temperature increment of 30 °C/h.
Electrophoretic Procedures--
Routinely, SDS-PAGE was carried
out as described by Laemmli (34). When testing for SDS resistance,
samples were solubilized in SDS sample buffer (final concentrations: 60 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 3%
-mercaptoethanol) and incubated at room temperature (not boiled) or
95 °C (boiled) for 5 min before analysis on a 15% polyacrylamide
gel. For analysis of the constituents of the protease-resistant core
complex, Tricine gel electrophoresis (16.5% T, 6% C) was used
(35).
Multi-angle Laser Light Scattering--
Size-exclusion
chromatography was performed on a HR-10/30 Superdex-200 column
(Amersham Pharmacia Biotech) in standard buffer containing 150 mM NaCl at a flow rate of 0.5 ml/min. The elution profiles
were monitored by UV absorption at 280 nm, light scattering at 632.8 nm, and differential refractometry. Light scattering and differential
refractometry were carried out using the Dawn and Optilab instruments,
respectively, of Wyatt Technology Corp. Analysis was carried out as
described by Astra software (36). For each sample, 100 µl of protein
solution (between 0.5 and 1 mg/ml protein) was loaded. The
dn/dc value (change of solution refractive index
with respect to a change in concentration of the molecules being
investigated) is fairly constant for proteins (37) and was set to 0.189 for the analysis of the light scattering data.
 |
RESULTS |
Biochemical and Biophysical Properties of Mixed SNARE
Complexes--
Four different SNARE proteins were examined for their
ability to form complexes with the SNAREs involved in neuronal
exocytosis. These included syntaxin 2, syntaxin 3, and syntaxin 4, three relatives of syntaxin 1 that exhibit a similar domain structure
(38), and are significantly homologous within the SNARE motifs (Fig. 1A). The homology is not
limited to the amino acids participating in core interactions but
includes residues on the surface. Despite their differential tissue
distribution and intracellular localization, these syntaxins are
probably involved in fusions of transport vesicles with the plasma
membrane and thus may interact physiologically with the neuronal SNAREs
in certain cell types. As the fourth example, we chose the R-SNARE
endobrevin/VAMP 8, a distant relative of synaptobrevin, which is
localized to endosomal compartments (26, 27). Within the SNARE motif,
only the amino acids of the core layers are partially conserved, with
much less similarity in the rest of the sequence (Fig.
1A).

View larger version (50K):
[in this window]
[in a new window]
|
Fig. 1.
Formation of SDS-resistant complexes between
various SNARE proteins. A, sequence alignment of the subset
of syntaxin and synaptobrevin homologs used for complex formation.
Alignment is restricted to the region encompassing the 16 interacting
layers of the synaptic fusion complex. The residues participating in
the layers are indicated by arrows and numbered
as in Refs. 24 and 25. Identical and conserved amino acids are
darkly and lightly shaded,
respectively. GenBankTM accession numbers are as follows:
synaptobrevin 2 (M24105), endobrevin/VAMP 8 (pending), syntaxin 1A
(D45208), syntaxin 2 (L20823), syntaxin 3 (L20820), and syntaxin 4 (L20821). B, approximately equal molar ratios of purified
SNARE proteins were mixed, incubated overnight, and subjected to
SDS-PAGE without boiling of the sample. After the run, the gel was
stained with Coomassie Blue. Synaptobrevin forms SDS-resistant
complexes with syntaxin 1-4 (SX1, 2,
3, and 4, respectively), whereas endobrevin only
forms SDS-resistant complexes with syntaxin 1 and 2.
|
|
For the binding experiments, all proteins were expressed in
Escherichia coli and purified (see "Experimental
Procedures" for details). The proteins were mixed in various
combinations and then analyzed by SDS-PAGE for the formation of
SDS-resistant complexes. It was shown previously that the synaptic
SNARE complex is resistant to SDS, a feature widely used for monitoring
complex formation (39). Of the eight SNARE combinations, six formed
SDS-resistant complexes, as demonstrated by the appearance of protein
bands with apparent molecular masses corresponding to ternary complexes (Fig. 1B). No SDS-resistant complex was observed with the
combinations SNAP-25/endobrevin/syntaxin 3 and syntaxin 4, respectively. However, complex formation was detected when the samples
were analyzed by non-denaturing PAGE or size exclusion chromatography
(data not shown, see also below).
For further analysis, we chose three representative complexes including
endobrevin/syntaxin 1/SNAP-25, synaptobrevin 2/syntaxin 4/SNAP-25, and
endobrevin/syntaxin 3/SNAP-25. These complexes were purified by ion
exchange chromatography and then subjected to limited proteolysis.
Previously, it was shown that the core domain of the synaptic SNARE
complex, i.e. the region of the interacting SNARE motifs, is
protease-resistant, whereas non-interacting SNARE motifs are
efficiently cleaved (16, 17). As shown in Fig. 2a, digestion of all three
complexes with proteinase K yielded a fragment migrating at about 16 kDa and a group of bands migrating between 8 and 14 kDa. When the
digests of the endobrevin/syntaxin 1/SNAP-25 and synaptobrevin
2/syntaxin 4/SNAP-25 complexes were not heated prior to
electrophoresis, a single SDS-resistant band was visible instead of the
group of 8-14-kDa bands whereas the 16-kDa band remained unchanged
(Fig. 2B). This result precisely corresponds to the
observations made previously for the synaptic SNARE complex (16) and
indicates that the 16-kDa represents the NH2-terminal
domain of the respective syntaxins, whereas the 8-14-kDa bands
represent the complex-forming SNARE motifs. These findings suggest that
protease-resistant core domains are formed that are very similar to
that of the synaptic complex.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 2.
Limited proteolysis reveals a similar domain
structure of the non-cognate SNARE complexes to the neuronal SNARE
complex. The following non-cognate SNARE complexes were purified
and subjected to limited proteolysis by proteinase K:
endobrevin/syntaxin1/SNAP-25 (EB/SX1/SN25), synaptobrevin
2/syntaxin 4/SNAP-25 (SB2/SX4/SN25), and endobrevin/syntaxin
3/SNAP-25 (EB/SX3/SN25). A, after digest all
samples were boiled in SDS-containing sample buffer and analyzed by the
Tricine variant of SDS-PAGE (35) followed by Coomassie Blue staining.
The positions of the NH2-terminal domains of the respective
syntaxins, and of the fragments contributing to the core domains, are
indicated. B, same as in A but without boiling of
the sample. An SDS-resistant core SNARE domain is separated from a
proteolytic fragment of the NH2-terminal region of the
respective syntaxin as indicated. C, after proteolysis, the
complexes endobrevin/syntaxin 3/SNAP-25 (EB/SX3/SN25), which
does not form an SDS-resistant complex, and synaptobrevin 2/syntaxin
4/SNAP-25 (SB2/SX4/SN25) were objected to size exclusion
chromatography on a Superdex 200 column. Fractions containing
proteolytic fragments were separated by Tricine gel electrophoresis.
For both complexes, the core SNARE domain consisting of several small
proteolytic fragments with an apparent molecular mass between 14 and 8 kDa is separated from the NH2-terminal domain of the
respective syntaxin.
|
|
No such SDS-resistant band containing the 8-14-kDa SNARE motifs was
observed with the endobrevin/syntaxin 3/SNAP-25 complex (data not
shown). Therefore, we have analyzed the digest of this complex by
size-exclusion chromatography. Fig. 2C shows that a single
major peak eluted from the column which contained a group of 8-14-kDa
bands (presumably representing the core complex forming SNARE motifs).
The 16-kDa band (presumably representing the NH2-terminal domain of syntaxin 3) was well separated and eluted at a position corresponding to a smaller molecular mass (Fig. 2C). The
first peak eluting from the column was further analyzed by MALLS, a procedure allowing for a direct determination of the molecular mass
irrespective of the shape of the complex (36). The procedure resulted
in a molecular mass of 41.3 (± 0.7) kDa, which is similar to that of
the core of the synaptic SNARE complex (16). A similar elution profile
was obtained when the digest of the SDS-resistant synaptobrevin
2/syntaxin 4/SNAP-25 complex was separated (Fig. 2C). This
indicates that these complexes contain the four SNARE motifs in a
1:1:1:1 stoichiometry.
As outlined above, the synaptic complex is represented by an extended
bundle of four
-helices yielding a characteristic
-helical spectrum in CD measurements. CD-spectroscopy of the three non-cognate complexes analyzed here resulted in similar spectra (data not shown),
further suggesting that the structure of all complexes is very similar.
Using CD spectroscopy as a means to monitor unfolding, we next examined
the thermal stability of the complexes. Previous work has shown that
the synaptic complex is remarkably resistant to thermal denaturation, a
feature believed to be a hallmark of SNARE complexes (21). As shown in
Fig. 3, the thermal denaturation curves
of the three complexes are virtually superimposable. All three
complexes are almost as stable as the neuronal complex (Fig. 3) and
clearly more stable than the exocytotic SNARE complex of Saccharomyces cerevisiae (22). Together these data show that the features of all complexes are very similar and suggest that differential sensitivities to SDS do not reflect major differences in
the biochemical and biophysical properties.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 3.
Thermal stability of SNARE complexes.
The following SNARE complexes were purified by ion exchange
chromatography: synaptobrevin 2/syntaxin 1/SNAP-25
(SB2/SX1/SN25), endobrevin/syntaxin 1/SNAP-25
(EB/SX1/SN25), synaptobrevin 2/syntaxin 4/SNAP-25
(SB2/SX4/SN25), and endobrevin/syntaxin 3/SNAP-25
(EB/SX3/SN25). Thermal denaturation of these complexes was
monitored by circular dichroism (CD) spectroscopy. The
change in the mean residue ellipticity [ ] at 220 nm of the
purified SNARE complexes was measured in standard buffer containing 100 mM NaCl.
|
|
Disassembly of Mixed SNARE Complexes--
The structural
similarities between the various SNARE complexes prompted us to
investigate whether these complexes are "functional" with respect
to the action of the disassembly chaperone NSF. With a few specialized
exceptions, NSF is thought to operate on all SNARE complexes (1, 15).
NSF binds to SNARE complexes in the presence of
-SNAP and ATP. When
ATP hydrolysis is permitted, the complexes reversibly disassemble into
their monomeric constituents. This reaction is currently thought to be
responsible for the regeneration of active SNAREs after fusion is complete.
Purified SNARE complexes were incubated in the presence of recombinant
NSF and
-SNAP under conditions either allowing or prohibiting ATP
hydrolysis by the ATPase NSF. To measure disassembly, we monitored the
cleavage of SNAP-25 by the light chain of botulinum neurotoxin E
(BoNT/E). Botulinum neurotoxins cleave the neuronal SNAREs only in the
disassembled state, whereas the ternary complex is toxin-resistant
(39-42). Fig. 4 (lanes
1) shows that not only the neuronal complex (left
panel) but also the heterologous SNARE complexes are
resistant to BoNT/E. When ATP hydrolysis by NSF was permitted, SNAP-25
was efficiently cleaved in each case (Fig. 4, lanes 3),
demonstrating that all complexes can be disassembled by NSF and
-SNAP.

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 4.
Disassembly of SNARE complexes. The
purified SNARE complexes (see Fig. 3) were incubated with BoNT/E alone
(lanes 1), or with BoNT/E, NSF, and -SNAP, in
the presence of EDTA (lanes 2) or
Mg2+ (lanes 3). NSF-driven
disassembly rendered SNAP-25 susceptible to cleavage by the BoNT/E
light chain that was visualized by Tricine gel electrophoresis and
immunoblotting using the monoclonal antibody Cl 71.1 (33).
|
|
Further Characterization of the Non-cognate Endobrevin/Syntaxin
1/SNAP-25 Complex--
In the last series of experiments, we
investigated whether structural and kinetic properties of the assembly
reaction are changed when synaptobrevin is replaced by its distant
relative endobrevin in the synaptic complex. The cognate SNARE partners of endobrevin are not yet known, but its localization and its tissue
distribution make it highly unlikely that it interacts with the
synaptic SNAREs in intact cells. To confirm that endobrevin does not
form complexes with the neuronal SNAREs, endobrevin and synaptobrevin 2 were coimmunoprecipitated from detergent extracts of PC12 cells. As
shown in Fig. 5, syntaxin 1 coprecipitated with synaptobrevin 2 but not with endobrevin even though
precipitation of endobrevin was almost quantitative.

View larger version (58K):
[in this window]
[in a new window]
|
Fig. 5.
Endobrevin does not form a complex with
syntaxin 1 in PC12 cells. Immunoblot analysis of
immunoprecipitations from Triton X-100 solubilized PC12 cells using an
affinity-purified polyclonal antibody for endobrevin (EB) or
the 69.1 (28) monoclonal antibody for synaptobrevin 2 (SB). The immunoprecipitate and supernatant were analyzed
with the endobrevin and synaptobrevin antibodies used for precipitation
and the monoclonal HPC-1 antibody for syntaxin 1 (30). h.c.
and l.c. indicate the positions of the IgG heavy and light
chains, respectively.
|
|
As in our previous work (16, 20, 21), we used CD spectroscopy to
monitor structural changes during assembly. We had shown before that
synaptobrevin is unfolded as a monomer but assumes an
-helical
conformation upon assembly (21). Similarly, the CD spectrum of
monomeric endobrevin was typical for unfolded proteins (data not shown)
(43). However, a large increase in
-helical content is observed upon
formation of the endobrevin/syntaxin 1/SNAP-25 complex (Fig.
6), which is comparable to that observed during the formation of the synaptobrevin/syntaxin 1/SNAP-25 complex (21).

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 6.
Structural changes upon formation of a
non-cognate SNARE complex between endobrevin, SNAP-25, and syntaxin
1. Upon mixing of endobrevin with the neuronal SNAREs SNAP-25, and
syntaxin 1, a major increase in -helical content was observed.
Dotted lines represent the theoretically
noninteracting mean residue ellipticities calculated from the observed
spectra of the individual proteins. The CD spectrum of the combined
components was measured in standard buffer containing 100 mM NaCl at 25 °C after overnight incubation of the
proteins.
|
|
These data show that the assembly of the endobrevin complex involves
structural changes remarkably similar to that of the genuine synaptic
SNARE complex. However, despite these structural and thermodynamic
similarities, it cannot be ruled out that there is a kinetic preference
for the formation of the native complex. To test for this possibility,
we monitored formation of ternary complexes in the presence of about
equal concentrations of endobrevin and synaptobrevin. The syntaxin
1/SNAP-25 binary complex was purified and incubated either with
synaptobrevin alone, endobrevin alone, or with a mix of both proteins.
Parallel experiments were carried out in which only the SNARE motif of
syntaxin (SX180-262) was present in the binary complex with SNAP-25.
Complex formation was monitored by the appearance of SDS-resistant
complexes, which were distinguishable due to their different apparent
molecular masses. As shown in Fig. 7,
about equal amounts of each of the ternary complexes formed when
synaptobrevin and endobrevin were present, demonstrating that there is
no kinetic preference for the cognate versus the non-cognate
R-SNARE regardless of whether intact or truncated syntaxin was
used.

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 7.
Endobrevin and synaptobrevin 2 are equally
efficient in binding to the synaptic SNAREs syntaxin 1 and
SNAP-25. Purified binary complexes consisting of SNAP-25 and
either the whole cytoplasmic region of syntaxin (SX1-265)
or a fragment of syntaxin containing the SNARE motif
(SX180-262) (16, 21) were incubated with either the
R-SNAREs synaptobrevin (SB), endobrevin (EB), or
a mixture of both at about equal concentrations. After 2 h of
incubation at 25 °C, the samples were analyzed by SDS-PAGE with
(lower panel) or without (upper
panel) boiling in SDS sample buffer followed by Coomassie
Blue staining. Both R-SNAREs were able to form SDS-resistant ternary
SNARE complexes. When incubated simultaneously, about equal amounts of
SDS-resistant SNARE complexes containing either synapto- or endobrevin
were formed. Note that, due to carryover of residual thrombin in the
endobrevin-containing sample, the His6 tags of
synaptobrevin, SX180-262, and SNAP-25 were cleaved, giving rise to
band multiplicity. The thrombin-cleaved band of the syntaxin fragment
is indicated by SX180-262 (th).
|
|
 |
DISCUSSION |
In the present study, we have shown that complex formation between
SNARE proteins is less specific than previously assumed (1, 44). Using
four different syntaxins and a distant relative of synaptobrevin, we
found that promiscuous SNARE complexes can be formed in arbitrary
combination. The features of these complexes are remarkably similar to
those of the genuine synaptic complex with respect to assembly,
disassembly, and biophysical properties, strongly suggesting that they
are, at least in vitro, functionally interchangeable.
The crystal structure of the core domain of the synaptic SNARE complex
allowed the identification of layers of interacting amino acids in the
core of the four helix bundle (24). The data presented here strongly
support the view that these interactions are indeed essential in
defining the features of SNARE complexes. Modeling showed previously
that syntaxin 1 can be replaced with syntaxin 4 without major steric
and electrostatic penalties (25), a hypothesis now supported by
experimental evidence. However, the degree of promiscuity in complex
formation between distantly related SNAREs was surprising. The sequence
identity between endobrevin and synaptobrevin is low (33%) (26, 27),
but the amino acids forming the core layers are either identical or at
least similar (Fig. 1A).
Several conclusions can be drawn from these observations. First, it is
becoming clear that the amino acids in the core are the essential
residues for SNARE complex formation with the residues on the surface
of the complex being less important. Second, the features of these
complexes are virtually indistinguishable with respect to domain
structure, stability, conformational change, and disassembly. These
remarkable similarities indicate that at least the complexes
investigated here form four-helix bundles supporting our previous
hypothesis that all SNARE complexes exhibit this basic structure (25).
Apparently, "drifts" in these features were not tolerated during
evolution, even though overall sequences are highly variable. This
lends strong support to the idea that it is these features that are
required for function, in full agreement with the current "zipper"
model of SNARE function in membrane fusion (3). We conclude that no
intrinsic property prevents "false" SNAREs from forming complexes
with each other, disproving one of the original tenets of the SNARE
hypothesis (1).
These arguments suggest that one needs to look elsewhere in order to
explain the indisputable specificity in SNARE interactions. As outlined
in the Introduction, there is evidence that individual SNAREs
participate in multiple interactions in yeast (14, 15). However, SNAREs
are remarkably specific with respect to their subcellular localization
(27, 45). Unfortunately, there is presently no reliable biochemical
method for discriminating cognate from non-cognate SNARE complexes,
although lack of co-immunoprecipitation is generally regarded as
evidence for non-interacting SNAREs. We were unable to coprecipitate
syntaxin 1 together with endobrevin from PC12 cells, i.e. a
cell line in which both the synaptic SNAREs and endobrevin are highly
expressed. This supports the view that they do not interact with each
other in intact cells. Despite localization to different subcellular
compartments, however, such non-cognate SNAREs may pass through the
same organelle during membrane recycling. Thus, sorting to different
compartments is probably insufficient to prevent non-cognate SNAREs
from forming complexes. Rather, control proteins must exist that
regulate individual SNAREs with a higher degree of specificity than
they display among each other. Such regulators may involve one of the
many additional proteins that specifically interact with individual
SNARE proteins, e.g. the Munc18/Sec1p protein family that
binds to the NH2-terminal domain of syntaxin, which, as
recently suggested (46) and further confirmed here, appears to be a
separately folded domain in the syntaxins 1-4.
In addition, the surface of individual SNARE complexes may carry
information that is selectively recognized by specific regulatory proteins. For instance, proteins such as synaptotagmin or complexin have been shown to interact with the synaptic SNARE complex, but it is
not yet known whether they would also bind to any of our non-cognate
complexes. The role of proteins binding to fully assembled SNARE
complexes remains to be elucidated. They may regulate SNARE function at
a late step in the fusion reaction in a positive or negative manner.
Discrimination between individual SNARE complexes would ensure that
regulation by such proteins is highly specific.