From the
The N-ethylmaleimide-sensitive fusion protein (NSF)
plays an essential role in intracellular membrane fusion events and has
been implicated in the exocytosis of synaptic vesicles. NSF binds
through soluble NSF attachment proteins (SNAPs) to a complex of
neuronal membrane proteins comprised of synaptobrevin, syntaxin, and
SNAP-25. Disassembly of this complex by NSF is thought to be a critical
step in the molecular events which lead to vesicle fusion with the
plasma membrane. Here we have studied the interaction of
Stimulated release of neurotransmitter from synaptic vesicles is
the fundamental process responsible for intercellular communication
within the nervous system. In the last few years, major advances have
been made in our understanding of synaptic vesicle docking and
exocytosis. There is now compelling evidence that neuronal exocytosis
operates by the same general mechanism as other intracellular membrane
fusion events, with the addition of a specialized regulatory mechanism
that enables Ca
Studies of vesicular transport between purified Golgi stacks by
Rothman and colleagues (3) led to the isolation and
identification of soluble proteins necessary for intracellular membrane
fusion. These include the N-ethylmaleimide-sensitive fusion
protein (NSF)
A set of neuronal SNAP
receptors was first identified by Rothman and colleagues(14) .
These receptors form a complex that contains the synaptic proteins
synaptobrevin (also known as VAMP), syntaxin, and SNAP-25 (synaptosomal
associated protein of 25 kDa)(14) . Synaptobrevin is a small
integral membrane protein with a single carboxyl-terminal transmembrane
domain found at high levels on synaptic
vesicles(15, 16) . Syntaxin is likewise an abundant
small integral membrane protein with a carboxyl-terminal transmembrane
domain but is localized primarily on the plasma
membrane(17, 18, 19) . SNAP-25 is palmitoylated
on a number of cysteine residues and is also found predominantly on the
plasma membrane(19, 20) . The observation that selective
cleavage of each of these three proteins by clostridial neurotoxins
leads to a complete blockade in neurotransmission provides strong
evidence that they are indeed essential in neuronal
exocytosis(21, 22, 23, 54) . There are
families of proteins related to synaptobrevin and syntaxin expressed
outside of the nervous system with varying subcellular distribution;
these related proteins are likely to serve as SNAP receptors for other
vesicular fusion events(24, 25) . Further evidence for
involvement of these protein families in membrane trafficking has been
provided by studies in yeast, where relatives of synaptobrevin,
syntaxin, and SNAP-25 have been shown to be essential at several stages
in the secretory pathway(2) .
Little is known about how NSF,
SNAPs, and their membrane receptors actually promote membrane fusion.
Some clues, however, have been provided by characterization of the
protein-protein interactions between them. In detergent extracts of
brain membranes, synaptobrevin, syntaxin, and SNAP-25 bind to each
other, forming the 7 S SNAP receptor complex(26) . This SNARE
complex binds
NSF catalyzes dissociation of the 20 S SNARE protein
complex in the presence of ATP. In this reaction it releases itself as
well as synaptobrevin, SNAP-25 and some
We have now studied the interactions between
Two vector systems were used to generate fusion
proteins in Escherichia coli. Expression in the vector pGEX-2T
(Pharmacia LKB Biotechnol.) generates fusion proteins with glutathione S-transferase (GST). Expression in the pTrcHis vector
(InVitrogen, Portland, OR) generates fusion proteins in which a
6-histidine tag and a 32-45-amino-acid linker sequence are fused
to the amino terminus. Full-length
Full-length syntaxin 1A in pGEX-2T
and pTrcHis has been previously described(34) .
Syntaxin 1A
fragments (residues 1-193 and 194-288) were introduced into
pGEX-2T after amplification with the following primers:
syntaxin(1-193): (sense, EcoRI)
5`-TATGAATTCTTATGAAGGACCGAACCCAG-3` (antisense, EcoRI)
5`-GCGAATTCCTAACTGAGGGCCTGCTTCGA-3`; syntaxin(194-288): (sense, EcoRI) 5`-CGTGAATTCGGATGGAGATCGAGACCAGGCAC-3` (antisense, EcoRI) 5`-ATA-GAATTCCTATCCAAAGATGCCCCCG-3`.
SNAP-25B in
pTrcHisA has been previously described (35) as has synaptobrevin
II in pGEX-2T(36) . Synaptobrevin II was also introduced into
pTrcHisA as a BamHI-EcoRI fragment.
Fusion proteins expressed in pTrcHis were also purified essentially
as described previously(34) . Proteins were eluted in steps from
Ni
His
When syntaxin was used in
the assay,
In contrast to its binding to
syntaxin and SNAP-25,
Syntaxin was incubated with
Virtually
identical results were obtained when the vesicle protein synaptobrevin
was added to the experiment. In this case, syntaxin was prebound to
How stable is the
conformational change in syntaxin induced by NSF? Non-binding syntaxin*
was prepared using
NSF is an essential protein in numerous intracellular
membrane fusion reactions(3) . Using detergent extracts from
brain, Rothman and colleagues (14, 26) have demonstrated
that
Current models suggest that vesicles need to dock
on their target membrane, resulting in the assembly of the SNARE
complex, before SNAPs can bind and NSF can act. However, our data
demonstrate that purified syntaxin is sufficient to serve as an
effective binding partner for
The
carboxyl-terminal domain of syntaxin responsible for
Although the carboxyl-terminal domain of
syntaxin contains the binding site for
The finding that syntaxin alone
can serve as a receptor for
We wish to thank S. W. Whiteheart and J. E. Rothman
for cDNAs encoding His
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-SNAP and
NSF with individual components of this complex and have identified
syntaxin as a primary substrate for NSF/
-SNAP. We find that
-SNAP binds directly to syntaxin 1A as well as weakly to SNAP-25,
while it does not bind to synaptobrevin II. NSF binds to syntaxin
through
-SNAP and in the presence of ATP catalyzes a
conformational rearrangement which abolishes binding of itself and
-SNAP. This reaction leads to the previously described disassembly
of the fusion complex, since synaptobrevin binding to syntaxin is also
reduced.
-SNAP binds to a carboxyl-terminal syntaxin fragment
(residues 194-288) that also binds synaptobrevin and SNAP-25.
However, NSF action on this syntaxin fragment has no effect on the
binding of
-SNAP or synaptobrevin. This suggests that the
conformational change normally induced by NSF in syntaxin depends on an
interaction between carboxyl- and amino-terminal domains of syntaxin.
to rapidly trigger
secretion(1, 2, 3) . Understanding which
proteins are involved in neuronal exocytosis and how they work together
to bring about membrane fusion is now the focus of considerable effort.
(
)and the soluble NSF attachment
proteins (SNAPs) which comprise a small family of homologous isoforms,
designated as
-,
-, and
-SNAP(4, 5, 6) . NSF and the SNAPs have
been shown to participate in most intracellular fusion reactions that
are amenable to in vitro analysis, and their homologs in yeast
are indispensable for vesicular transport through the secretory
pathway(2, 3, 7) . NSF is an ATPase which in the
presence of a SNAP promotes membrane fusion between uncoated transport
vesicles and their target
compartment(8, 9, 10) . NSF does not bind
directly to membranes but instead binds to
/
- and
-SNAP
which attach to specific membrane receptors (SNAP receptors or
SNAREs)(11, 12, 13) .
-SNAP and NSF to form a 20 S particle which is
stable in the presence of a non-hydrolyzable ATP
analog(12, 26) , and is thought to comprise the core of
a molecular membrane fusion machine. Since the SNARE complex contains
membrane proteins from both the vesicle (v-SNAREs) and the plasma
membrane (t-SNAREs), it has been proposed that selectivity in pairing
between v- and t-SNAREs may enable a transport vesicle to choose the
appropriate target site at which to dock and
fuse(26, 27) . The finding that certain isoforms of
syntaxin (syntaxin 1A and 4, but not syntaxin 2 or 3) preferentially
interact with synaptobrevin II (27) and that SNAP-25 selectively
potentiates these interactions (28) suggests that there is
selectivity in the pairing of v- and t-SNAREs as they assemble into a
SNARE complex.
-SNAP from
syntaxin(12, 14, 26) . This disassembly precedes
membrane fusion, since assembled SNARE complexes accumulate in yeast
secretory mutants deficient in sec18 (NSF) activity(29) . The
ATPase activity of NSF is essential for its role in promoting membrane
fusion, since site-directed mutants with impaired ATPase activity do
not support Golgi transport in vitro(10, 30) .
Whether disassembly of the SNARE complex by NSF leads directly to
membrane fusion, or serves as a priming step for a subsequent, perhaps
Ca
stimulated reaction that causes membrane fusion is
unknown. Furthermore, exactly how the SNAPs and NSF interact with the
individual components of the SNARE complex remains an open question.
-SNAP, NSF, and
purified individual synaptic SNARE proteins (syntaxin, SNAP-25, and
synaptobrevin). We find that syntaxin binds with moderate affinity to
-SNAP and that the syntaxin-
-SNAP complex is a target for NSF
action. NSF uses the energy derived from ATP hydrolysis to induce a
conformational change through
-SNAP in syntaxin. This reaction
leads to the previously described disassembly of the synaptic fusion
complex.
Plasmid Construction
Plasmids encoding
His-
-SNAP, untagged
-SNAP(6) , and
His
-NSF (31) were kindly provided by S. W.
Whiteheart and J. E. Rothman (New York). cDNAs encoding rat syntaxin
1A(17) , synaptobrevin II(15) , and SNAP-25B (22, 32) were generously provided by R. H. Scheller
(Stanford University) and T. C. Südhof (Dallas), respectively.
Full-length and truncated coding sequences were amplified using the
polymerase chain reaction and oligonucleotides containing appropriate
restriction sites for subcloning into the indicated
plasmid(33) .
- and
-SNAP and two
fragments of
-SNAP (residues 1-154 and 114-295) were
amplified using polymerase chain reaction and were subcloned into
pGEX-2T. Primers used for amplification included:
-SNAP
full-length: (sense, EcoRI) 5`-TATAGGATCCATGGACAACTCCGGGAAG-3`
(antisense, EcoRI) 5`-ATAGAATTCTTAGCGCAGGTCTTCCTC-3`;
-SNAP(1-154): (sense, EcoRI)
5`-TATAGGATCCATGGACAACTCCGGGAAG-3` (antisense, EcoRI)
5`-GCAGAATTCGCCTTTGTAGTAGTCTGC-3`;
-SNAP(114-295): (sense, EcoRI) 5`-CGTGAATTCGTATGGGCCGCTTCACCATC-3` (antisense, EcoRI) 5`-ATAGAATTCTTAGCGCAGGTCTTCCTC-3`;
-SNAP: (sense, BamHI) 5`-GCGGATCCATGGACAACGCG-3` (antisense, BamHI)
5`-GCGGATCCTCACTTGAGGTCTCCGTC-3`.
Purification of Fusion Proteins
GST fusion
proteins were purified essentially as described (34, 36) except that HEPES-buffered saline (HBS: 20
mM HEPES, pH 7.4, 150 mM NaCl, 1 mM
dithiothreitol) was used as the primary buffer. In the purification of
proteins retaining their transmembrane domains (full-length syntaxin,
synaptobrevin; syntaxin(194-288)) 0.5% Triton X-100 was added to
all washing buffers. Synaptobrevin II was separated from GST by
treating the glutathione-Sepharose-immobilized fusion protein with 1
unit of thrombin (Sigma) per 500 µl of beads in HBS containing 0.5%
Triton X-100 at room temperature for 1 h(37) . Thrombin was then
inhibited by addition of 1 mM phenylmethylsulfonyl fluoride,
and the supernatant containing released synaptobrevin was collected.
-Sepharose (ProBond, InVitrogen) with buffer
containing 80 mM, 160 mM, or 400 mM imidazole, pH 7.6. In a typical purification from 1 liter of
bacteria, 0.8 ml of resin was used and eluted with two 1-ml fractions
at each imidazole concentration. Fractions were analyzed for purity by
SDS-PAGE and staining with Coomassie Blue and then dialyzed against HBS
and, when appropriate, 0.5% Triton X-100. Syntaxin 1A(1-288) was
most concentrated in fractions eluted with 400 mM imidazole,
and a typical preparation from 1 liter of bacteria produced 0.5 mg of
essentially pure syntaxin. SNAP-25 was most concentrated in fractions
eluted with 160 mM imidazole, and a typical preparation from 1
liter of bacteria produced 100 µg of SNAP-25 which was 50-75%
pure. Synaptobrevin II was also most concentrated in fractions eluted
with 160 mM imidazole, and a typical preparation from 1 liter
of bacteria produced 200 µg of synaptobrevin which was 50-75%
pure.
-NSF and His
-
-SNAP (expressed
in the pQE9 vector (Qiagen)) were purified essentially according to
published procedures(6, 38) . His
-NSF was
eluted from Ni
-Sepharose with a continuous gradient
of imidazole (0-500 mM in 20 mM HEPES-KOH, pH
7.8, 200 mM KCl, 2 mM MgCl
, 10% glycerol,
1 mM ATP, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride). Aliquots were snap frozen and
stored at -70 °C. His
-
-SNAP was eluted from
Ni
-Sepharose with steps of 80 mM, 160
mM, and 400 mM imidazole as above. A typical
preparation of
-SNAP from 1 liter of bacteria produced >10 mg
of essentially pure protein.
Production of Antibodies
New monoclonal antibodies
recognizing - and
-SNAP were generated (Fig. 1).
Purified
-SNAP (cleaved with thrombin from a GST-
-SNAP fusion
protein) was used to immunize BALB/c mice. Two hybridoma cell lines
(clones Cl 77.1 and 77.2) were established using standard procedures
for fusion, propagation, and screening(39, 40) . A
polyclonal rabbit serum recognizing NSF (R32) was generated using
purified His
-NSF by standard procedures(41) .
Figure 1:
Characterization of monoclonal
antibodies specific for - and
-SNAP. A, immunoblots
of total rat brain homogenate were probed with two monoclonal
antibodies raised against recombinant
-SNAP. Each lane is an
immunoblot of S1 homogenate (15 µg) separated on a 12%
SDS-polyacrylamide gel. Ascites fluid containing antibody from clone Cl
77.1 or Cl 77.2 (1:5000 dilution) was used to immunodecorate the blots.
Proteins recognized were visualized using alkaline phosphatase coupled
secondary antibody reagents. B,
-SNAP and
-SNAP-GST
fusion proteins were expressed in bacteria and immobilized on
glutathione-Sepharose beads. An immunoblot of 2 µg of each fusion
protein is shown. GST alone is included as a
control.
Immunoprecipitations
Purified proteins were
incubated together at the concentrations indicated in a final volume of
100 µl at 4 °C with gentle agitation. Standard binding assay
buffer included 20 mM HEPES, pH 7.4, 150 mM NaCl, 1
mM dithiothreitol, and 0.5% Triton X-100. After 4 h, samples
were briefly spun to remove any aggregated material (5 min, 12,000
revolutions/min in 4 °C microfuge). After transfer to a fresh tube,
5 µl of ascites fluid (containing 5-15 µg of specific
IgG) was added, and incubations were continued for 1 h. 25 µl of a
70% protein G-Sepharose suspension was then added, and samples were
incubated for an additional 1 h. The beads were collected by brief
centrifugation (30 s, 3000 revolutions/min in microfuge), and then
washed three times with 1 ml of buffer. Proteins bound to the beads
were finally solubilized with 40 µl of 1.5 SDS sample
buffer (42) and heated for 5 min. The monoclonal antibodies used
for immunoprecipitation included HPC-1 for syntaxin(43) , Cl
69.1 for synaptobrevin(36) , and Cl 71.2 for SNAP-25 (35).
Binding to Glutathione-Sepharose Immobilized
Proteins
Soluble proteins were incubated together with the
indicated amount of GST fusion protein immobilized on
glutathione-Sepharose beads in standard binding buffer. Incubations
were for 4 h, after which the beads were washed and processed as
described for protein G-Sepharose beads above.
Disassembly Reactions
Syntaxin was incubated
together with -SNAP and, where indicated, synaptobrevin for 2 h in
a prebinding reaction. The buffer used was the same as in the binding
reactions above, but also contained 2 mM MgCl
.
Disassembly reactions were initiated by addition of NSF (2-5
µg/100 µl reaction) together with ATP or ATP
S (1
mM each), and allowed to proceed for 1 h. When immobilized
GST-syntaxin was used in the disassembly reaction, the beads were
washed directly after the reaction, and bound proteins were solublized
in 1.5
SDS sample buffer as above. When
His
-syntaxin was used in the disassembly reaction, it was
collected by immunoprecipitation with HPC-1 and protein G-Sepharose
beads, as described above.
Other Procedures
Samples were separated by
SDS-PAGE and transferred to nitrocellulose as described(34) .
Blots were incubated with monoclonal antibodies (ascites fluid) or
polyclonal sera diluted 1:1000-1:2000 in Blotto (20 mM Tris, pH 7.4, 150 mM NaCl, 5% powdered non-fat milk, and
0.1% Tween 20) overnight at 4 °C. Blots to be probed with I-protein A were washed and incubated with secondary
rabbit anti-mouse IgG antibodies (Cappel), washed again and then
incubated with 0.1 µCi/ml
I-protein A (DuPont NEN)
for 1-2 h, and finally washed five to six times over >2 h.
After drying, membranes were exposed to x-ray film. For quantitation,
radioactive membranes were exposed to Phosphor-screens which were
analyzed on a PhosphorImager (Molecular Dynamics) using the ImageQuant
software. When alkaline phosphatase-coupled secondary goat anti-mouse
or anti-rabbit antibodies were used, immunoreactive bands were
visualized by addition of nitro blue tetrazolium (0.33 mg/ml) and
5-bromo-4-chloro-3-indolyl phosphate (0.17 mg/ml). Densitometry (used
for relative quantitation of Coomassie-stained bands) was performed
using a Visage 2000 Scanner (BioImage Products, MilliGen/Biosearch
Division of Millipore, Ann Arbor, MI). Protein concentrations were
determined by the method of Bradford using bovine serum albumin as a
standard (44) or estimated from Coomassie Blue-stained gels of
recombinant proteins.
Production and Characterization of Antibodies
Recognizing
To study the interaction of -SNAP
-SNAP
with its receptors, we raised monoclonal antibodies directed against
recombinant
-SNAP. Two hybridoma cell lines were developed and are
designated Cl 77.1 and Cl 77.2. As shown in Fig. 1A,
each recognized two proteins in a rat brain homogenate with apparent
molecular masses of 33 and 34 kDa. Based on the high similarity between
-SNAP and
-SNAP (83% identity(6) ), it is likely that
this doublet consists of
- and
-SNAP. Using recombinant GST
fusion proteins encoding
-SNAP and
-SNAP, we confirmed this
specificity (Fig. 1B). Both antibodies recognize
-
and
-SNAP, and neither antibody recognizes GST alone.
In the first series of experiments, we asked
whether -SNAP Binds to Syntaxin and SNAP-25 but Not to
Synaptobrevin
-SNAP can bind to the individual SNARE proteins syntaxin,
SNAP-25, and synaptobrevin. For this purpose we expressed full-length
syntaxin 1A (referred to henceforth as syntaxin), synaptobrevin II
(referred to as synaptobrevin), SNAP-25, and
-SNAP each in
bacteria fused to either a 6-histidine (His
) or GST tag.
The proteins were purified by affinity chromatography (using
Ni
- or glutathione-Sepharose) and when indicated
separated from the GST moiety by thrombin cleavage. To assay for
binding, each SNARE protein was incubated separately with increasing
concentrations of
-SNAP. The SNARE proteins were then collected by
immunoprecipitation using specific monoclonal antibodies and analyzed
for bound
-SNAP by immunoblotting.
-SNAP bound efficiently (Fig. 2A), with
a 50% effective concentration (EC
) of between 500 nM and 1 µM. At saturation, about 1 mol of
-SNAP
was bound for every 3-4 mol of syntaxin collected, as judged by
densitometry of Coomassie Blue-stained samples (see, for example, Fig. 3A). Immunoprecipitation of
-SNAP by the
syntaxin-specific antibody HPC-1 was dependent on the presence of
syntaxin, as essentially no
-SNAP was detected in control
precipitations lacking syntaxin (not shown, but see Fig. 3A). This demonstrates that under these conditions
-SNAP did not adsorb significantly to the plastic surface of the
test tubes, as has been described when higher concentrations of
-SNAP are used (5, 13, 45). We also tested the effect of
increasing concentrations of sodium chloride on the binding of
-SNAP to syntaxin (Fig. 2B). The interaction
between these proteins was unaffected by 500 mM sodium
chloride and was reduced by higher sodium chloride concentrations.
-SNAP binding to syntaxin is thus relatively salt resistant, as is
the binding of synaptobrevin and SNAP-25 to syntaxin(46) .
Figure 2:
-SNAP binding to syntaxin 1A,
SNAP-25, and synaptobrevin II. A,
-SNAP binding to
syntaxin. His
-syntaxin 1A (300 nM) was incubated
with increasing concentrations of His
-
-SNAP (0-5
µM), and then collected by immunoprecipitation using a
syntaxin-specific monoclonal antibody (HPC-1) and protein G-Sepharose.
Precipitated proteins were visualized by SDS-PAGE and immunoblotting
for
-SNAP (Cl 77.2) or syntaxin (HPC-1). Bound antibody was
visualized using
I-protein A and autoradiography. Note
that equal amounts of syntaxin were recovered in each precipitation. B, effect of NaCl on
-SNAP binding to syntaxin.
His
-syntaxin (300 nM) was incubated with 5
µM His
-
-SNAP in the presence of
increasing concentrations of NaCl as indicated. Immunoprecipitation and
immunoblotting was carried out as in A. C,
-SNAP
binding to SNAP-25. His
-SNAP-25 (300 nM) was
incubated with increasing concentrations of His
-
-SNAP
(0-5 µM), and collected by immunoprecipitation using
the SNAP-25-specific monoclonal antibody Cl 71.2. Proteins were
visualized by immunoblotting as above using specific antibodies
(polyclonal rabbit SNAP-25 antibody, Cl 77.2 for
-SNAP). D, effect of NaCl on
-SNAP binding to SNAP25.
His
-SNAP-25 (300 nM) was incubated with 5
µM His
-
-SNAP in the presence of
increasing concentrations of NaCl as indicated. E,
-SNAP
binding to synaptobrevin. Synaptobrevin II (300 nM, prepared
by thrombin cleavage from GST-synaptobrevin fusion protein) was
incubated with
-SNAP (0.7-5 µM) and
immunoprecipitated using the synaptobrevin II-specific antibody (Cl
69.1) (36). Immunoprecipitates were subjected to immunoblotting in
parallel with the samples shown in A using antibodies
recognizing
-SNAP or synaptobrevin. The exposure shown is the same
as shown in A; even after a four times longer exposure no
-SNAP signal could be detected (not
shown).
Figure 3:
Binding of NSF to the -SNAP-syntaxin
complex and dissociation of the complex by ATP. A, effect of
NSF on the binary syntaxin-
-SNAP complex. Syntaxin (0.9
µM) was incubated with
-SNAP (5 µM) in a
buffer containing Mg
for 2 h prior to addition of NSF
(0.3 µM) together with ATP or ATP
S (1 mM) as
indicated. After an additional 30 min, the syntaxin was collected by
immunoprecipitation as in Fig. 2. The precipitated proteins were
separated by SDS-PAGE and visualized by protein staining with Coomassie
Blue. Note that
-SNAP was recovered in the immunoprecipitate only
when syntaxin was included (compare lanes 1 and 3).
NSF only bound to syntaxin in the presence of
-SNAP (compare lanes 5 and 7) and a non-hydrolyzable ATP analog
(compare lanes 7 and 8). B, effect of NSF on
synaptobrevin binding to syntaxin. A similar experiment to that shown
in A, except that each reaction contained synaptobrevin, and
the proteins recovered in the immunoprecipitates were detected by
immunoblotting. Syntaxin (0.5 µM), synaptobrevin (0.5
µM), and
-SNAP (5 µM) were preincubated
and then treated with NSF as above. Proteins collected in the washed
immunoprecipitate pellets were subjected to SDS-PAGE and
immunoblotting. Bound proteins were visualized using specific
antibodies (see ``Materials and Methods'') and
I-protein A followed by autoradiography, except for
synaptobrevin for which alkaline phosphatase-coupled secondary antibody
and color substrates were used.
SNAP-25 also bound -SNAP, but with lower affinity since the
binding was not saturated at the highest concentration of
-SNAP
tested (10 µM) (Fig. 2C and not shown).
Furthermore, the binding of
-SNAP to SNAP-25 was reduced by
increasing the sodium chloride concentration to only 300 mM (Fig. 2D).
-SNAP did not appreciably bind to
synaptobrevin (Fig. 2E). No
-SNAP was
immunoprecipitated together with synaptobrevin even when the
-SNAP
concentration was as high as 10 µM (Fig. 2E and not shown). To be sure that the antibody used for
immunoprecipitation (Cl 69.1(36) ) did not itself interfere with
an interaction between
-SNAP and synaptobrevin, we repeated the
experiment using a GST-
-SNAP fusion protein immobilized on
glutathione-Sepharose. We incubated immobilized GST-
-SNAP with up
to 10 µM synaptobrevin, and after washing did not detect
any specific binding between synaptobrevin and
-SNAP (data not
shown).
Interaction of NSF with the
As outlined in the Introduction, Rothman and
collaborators (26) have demonstrated that -SNAP-Syntaxin
Complex
-SNAP must bind
to its receptors on the membrane before NSF can bind and act.
Furthermore, ATP hydrolysis by NSF leads to a dissociation of the SNAP
receptor (SNARE) complex, with a release of synaptobrevin II, SNAP-25,
-SNAP and NSF from syntaxin(26) . Given our finding that
-SNAP can bind to syntaxin in the absence of other SNAREs, we
tested whether the purified syntaxin-
-SNAP complex alone could
bind NSF, and whether ATP hydrolysis can dissociate this complex.
-SNAP, NSF, or both in the presence
of either Mg
ATP or non-hydrolyzable
Mg
ATP
S. Syntaxin was then immunoprecipitated,
and the collected material was visualized by protein staining. As shown
in Fig. 3A,
-SNAP binding to syntaxin was not
affected by Mg
or nucleotide (Fig. 3A, lanes 3 and 4). NSF bound to the
-SNAP-syntaxin
complex in the presence of non-hydrolyzable ATP
S demonstrating
that syntaxin alone is a bona fide SNAP receptor which can
promote NSF binding to
-SNAP (compare Fig. 3A, lane 7 to lanes 5 and 6). However, when ATP
was present, neither
-SNAP nor NSF coprecipitated with syntaxin.
The lack of
-SNAP binding to syntaxin must be due to a significant
change in their affinity for each other because the
-SNAP
concentration in this experiment (5 µM) was sufficient to
saturate binding to syntaxin prior to the addition of NSF (see Fig. 2). The effect of ATP was abolished when the samples were
incubated in the presence of 1 mM NEM (data not shown), a
condition known to inactivate NSF(45, 47) . We conclude
that ATP hydrolysis by NSF caused dissociation of
-SNAP and NSF
from syntaxin in the absence of any other proteins. We refer to this
new, non-binding, conformation of syntaxin as syntaxin*.
-SNAP and synaptobrevin before NSF and ATP or ATP
S were added (Fig. 3B). Synaptobrevin coprecipitated with syntaxin (Fig. 3B, lane 1), although the binding was
substoichiometric at the concentrations used (0.5 µM synaptobrevin, 0.5 µM syntaxin), and bound
synaptobrevin was not readily seen by protein staining with Coomassie
Blue. Immunoprecipitated proteins were therefore visualized by
immunoblotting. The syntaxin-synaptobrevin interaction was unaffected
by binding of
-SNAP to syntaxin (Fig. 3B, compare lanes 1 and 3). However, when NSF dissociated the
-SNAP-syntaxin complex in the presence of ATP, synaptobrevin no
longer coprecipitated with syntaxin (Fig. 3B, compare lanes 7 and 8). Syntaxin* thus no longer binds well
to synaptobrevin. Parallel experiments using recombinant SNAP-25
suggested that its binding to syntaxin* was also decreased by NSF
action (data not shown).
(
)These findings suggest
that the conformational change induced by NSF and
-SNAP in
purified recombinant syntaxin is the same change previously shown to
disrupt v-SNARE-t-SNARE pairing in the complete SNARE complex isolated
from brain(26) . Furthermore, tests of our NSF and
-SNAP
proteins on SNARE complexes from brain detergent extracts demonstrated
that similar concentrations of each were required for effective
disassembly of the complex (data not shown).
-SNAP, NSF, and ATP as described above. After a
1-h incubation, the ATPase activity of NSF was inhibited by chelating
Mg
with EDTA (Fig. 4, lanes 3 and 4), and incubations were continued for an additional 1 or 2 h.
A control reaction in which ATP
S was present throughout was
included to measure maximal levels of
-SNAP and NSF binding to
syntaxin (lane 1), as was a reaction in which Mg
was not chelated so that NSF remained active throughout the
experiment (lane 2). Syntaxin was then collected from all
samples by immunoprecipitation. As shown in Fig. 4,
-SNAP
and NSF gradually rebound to syntaxin when the ATPase activity of NSF
was inhibited. This suggests that the syntaxin conformation induced by
NSF is only transiently stable, and in the absence of other factors or
ongoing NSF activity ``syntaxin*'' will relax back to its
original ``syntaxin'' conformation.
Figure 4:
Rebinding of NSF and -SNAP to
syntaxin after quenching of NSF activity. Syntaxin (500 nM)
was incubated with
-SNAP (1 µM), and NSF (0.3
µM) in the presence of either
Mg
ATP
S (lane 1) or
Mg
ATP (lanes 2-4). After the first
hour, EDTA was added (5 mM) to the samples shown in lanes
3 and 4. Samples in lanes 1-3 were
incubated for 1 h, and the syntaxin was then immunoprecipitated. The
sample in lane 4 was incubated for 2 h prior to starting the
immunoprecipitation. Immunoprecipitated proteins were visualized by
SDS-PAGE and immunoblotting, using
I-protein A for
detection.
The cytoplasmic portion of syntaxin 1A contains a
carboxyl-terminal domain (amino acid residues 194-265) which is
sufficient to allow synaptobrevin(27) , SNAP-25(34) , the
-SNAP Binds to a Carboxyl-terminal Fragment of
Syntaxin
1 subunit of the class B N-type Ca
channel(49) , and synaptotagmin
(
)to
bind. An amino-terminal fragment of syntaxin (residues 1-193)
also binds directly to the carboxyl-terminal domain (residues
194-265), suggesting an intramolecular association(27) .
To determine which part of syntaxin is responsible for the binding of
-SNAP, we generated deletion mutants of syntaxin fused to GST.
Full-length, amino-terminal (residues 1-193), and
carboxyl-terminal (residues 194-288) syntaxin-GST fusion proteins
were immobilized on glutathione-Sepharose and then incubated with
-SNAP. After extensive washing, bound
-SNAP was detected by
immunoblotting.
-SNAP bound to full-length GST-syntaxin but not to
control beads containing the GST protein alone (Fig. 5A).
-SNAP also bound efficiently to the
carboxyl-terminal syntaxin fragment and did not bind to the
amino-terminal fragment (Fig. 5A).
-SNAP therefore
binds to the same domain on syntaxin (residues 194-288) which has
previously been shown to interact with the SNARE proteins. Since most
work characterizing the interactions of purified recombinant syntaxin
with other proteins has been done using the cytoplasmic domain of
syntaxin (residues 1-265), we also tested the effect of deleting
the trans-membrane domain from syntaxin on
-SNAP binding. As shown
in Fig. 5A, removal of this short sequence reduced the
binding of
-SNAP to syntaxin. This is reminiscent of the effects
of this same deletion on the cleavage of syntaxin by the neurotoxin
BoNT/C1 (23) and suggests that the transmembrane domain is important
for maintaining syntaxin in a conformation permissive for binding some
of its partners.
Figure 5:
Domain mapping for -SNAP-syntaxin
binding and test of NSF activity on the
-SNAP-syntaxin(194-288) complex. A, binding of
-SNAP to GST fusion proteins encoding full-length syntaxin, an
amino-terminal fragment (syntaxin residues 1-193), a
carboxyl-terminal fragment (syntaxin residues 194-288), a
cytoplasmic domain fragment (syntaxin residues 1-265) and, as a
control, GST alone. GST fusion proteins (10 µg) were immobilized on
glutathione-Sepharose and incubated with
-SNAP (5
µM). After extensive washing the material bound to the
beads was subject to electrophoresis and immunoblotting for
-SNAP.
The bound
-SNAP was detected using the 77.2 antibody and
I-protein A. B, binding of
His
-syntaxin to GST fusion proteins encoding full-length
-SNAP, an amino-terminal fragment (
-SNAP residues
1-154), and a carboxyl-terminal fragment (
-SNAP residues
114-295). GST fusion proteins (20 µg) immobilized on
glutathione Sepharose were incubated with His
-syntaxin (2.5
µM). Bound material was collected and visualized as in A using the syntaxin-specific monoclonal antibody HPC-1. C, effect of NSF activity on
-SNAP and synaptobrevin
binding to syntaxin GST fusion proteins. Full-length (1-288)
syntaxin or its carboxyl-terminal fragment (194-288) immobilized
on glutathione-Sepharose was incubated with synaptobrevin and
-SNAP. After a pre-binding period (2 h, 4 °C) NSF and either
Mg-ATP
S or Mg-ATP was added as shown. After 30 min, the beads were
washed extensively and proteins remaining bound visualized by
electrophoresis and immunoblotting.
In parallel experiments, we tested two fragments of
-SNAP for their ability to bind syntaxin (Fig. 5B).
Neither an amino-terminal (residues 1-154) nor a
carboxyl-terminal (residues 114-295)
-SNAP fragment fused to
GST was able to bind syntaxin. Syntaxin did bind tightly to full-length
-SNAP-GST. An intact
-SNAP appears therefore to be important
for the binding of syntaxin, and we cannot define a subdomain
responsible for syntaxin binding.
NSF Fails to Disassemble
We next
investigated whether the carboxyl-terminal domain of syntaxin bound to
-SNAP and Synaptobrevin
from the Syntaxin Carboxyl-terminal Fragment
-SNAP will allow NSF to bind and disassemble the complex (Fig. 5C). For this experiment, equal amounts of
full-length syntaxin or the carboxyl-terminal fragment (syntaxin
residues 199-288), expressed as GST-fusion proteins, were
immobilized on glutathione-Sepharose. The beads were preincubated with
synaptobrevin and
-SNAP, and NSF was then added together with
either ATP
S or ATP. After a 1-h reaction, the beads were washed,
and bound material was analyzed by immunoblotting. When full-length
GST-syntaxin was used, NSF was able to dissociate
-SNAP and
synaptobrevin, in agreement with results described above. When the
carboxyl-terminal fragment of syntaxin was used, synaptobrevin,
-SNAP, and NSF all bound. However, no dissociation of
-SNAP
or synaptobrevin was observed in the presence of ATP. The binding of
NSF itself to
-SNAP and syntaxin was reduced in the presence of
ATP, suggesting that it induced a change in
-SNAP which had no
effect on the syntaxin fragment. This suggests that while the
carboxyl-terminal fragment contains the sequences necessary for binding
-SNAP, as well as synaptobrevin and SNAP-25, it is not able to
assume the syntaxin* conformation induced by NSF.
-SNAP and NSF bind to an assembled complex of membrane
proteins consisting of synaptobrevin, syntaxin, and SNAP-25. NSF drives
an ATP-dependent disassembly of this complex, and this event is thought
to be intimately associated with membrane fusion(26) . We have
shown here that NSF and
-SNAP interact with purified syntaxin and
that NSF retains its ability to induce a conformational change in
syntaxin in this simplified system. This conformational change leads to
formation of what we refer to as syntaxin*, which no longer binds well
to
-SNAP or the other SNARE complex proteins. Formation of
syntaxin* and the resulting disassembly of the SNARE complex may lead
directly to membrane fusion as previously proposed(26) , or may
instead serve as an essential priming step which prepares syntaxin and
the other SNARE proteins for a downstream, possibly Ca
triggered, interaction leading to fusion. Essential features of a
model based on our results are depicted schematically in Fig. 6.
Figure 6:
Model showing the effect of NSF and
-SNAP on syntaxin. Syntaxin is shown on the presynaptic plasma
membrane before and after the conformational change catalyzed by NSF.
Filled stretches of syntaxin (two near the amino terminus, and one
toward the carboxyl terminus) are regions predicted to participate in
-helical coiled-coil interactions. Syntaxin can bind SNAP-25 on
the presynaptic plasma membrane and synaptobrevin on an incoming
synaptic vesicle to form a fully assembled SNAP receptor complex.
Binding of SNAP-25 and synaptobrevin is mediated by syntaxin sequences
located between amino acid residues 194 and 265, and is predicted to
depend on
-helical coiled coil interactions (34).
-SNAP
likewise binds to this carboxyl-terminal domain of syntaxin and can
bind in either the absence or presence of SNAP-25 and synaptobrevin.
NSF binds to the
-SNAP-syntaxin complex, again in either the
absence or presence of SNAP-25 and synaptobrevin. The
NSF-
-SNAP-syntaxin complex remains stable in the absence of
hydrolyzable ATP (i.e. with ATP
S or EDTA). Addition of
ATP and its hydrolysis by NSF leads via
-SNAP to a conformational
change in syntaxin. Syntaxin after NSF action, designated syntaxin*, no
longer binds
-SNAP, synaptobrevin, or SNAP-25. As shown, this is
likely a result of binding of the amino-terminal coiled-coil domains of
syntaxin to its own carboxyl-terminal domain. These intramolecular
interactions preclude binding of other proteins to the
carboxyl-terminal domain. Induction of syntaxin* and the resulting
dissociation of synaptobrevin and SNAP-25 may lead directly to membrane
fusion, or may be a priming step which prepares the molecules for a
subsequent fusion triggering reaction.
The t-SNAREs syntaxin 1A and SNAP-25 each bind specifically to
-SNAP and are therefore bona fide SNAP receptors, while
the v-SNARE synaptobrevin II does not directly bind
-SNAP (Fig. 2). Syntaxin binds
-SNAP with significantly higher
affinity than SNAP-25 does, suggesting that syntaxin is likely to be
the predominant
-SNAP receptor.
-SNAP may in fact bind
simultaneously to syntaxin and SNAP-25 in the neuronal plasma membrane,
since these two proteins are largely co-localized and are complexed to
each other(19) . This is particularly likely since
-SNAP
binds to the same domain of syntaxin which binds SNAP-25 (amino acids
194-288; Fig. 5), and also binds to the same domain of
SNAP-25 which binds syntaxin (amino acids 1-100).
(
)
-SNAP. Furthermore, NSF binds
specifically to the
-SNAP-syntaxin complex, and ATP hydrolysis
leads to the disassembly of this ternary complex in the absence of
other proteins (Fig. 3A). We believe that disassembly of
the NSF-
-SNAP-syntaxin complex is primarily due to a major
conformational change in syntaxin, which is induced by NSF and
transmitted through
-SNAP, for the following reasons. First,
syntaxin also loses affinity for synaptobrevin (Fig. 3B). Since synaptobrevin does not interact with
-SNAP directly, this suggests that a conformational change has
been induced in syntaxin which interferes with its ability to bind
synaptobrevin. Second, no disassembly of
-SNAP or synaptobrevin
could be induced from the carboxyl-terminal fragment of syntaxin,
despite efficient binding of
-SNAP and NSF and clear evidence of
NSF activity (Fig. 5C). This is difficult to explain if
NSF only affects
-SNAP or synaptobrevin. Finally, one would expect
that all free
-SNAP (which does not interact with NSF unless it is
bound to its membrane receptor) would have to be shifted to a
non-binding conformation if a change in
-SNAP would be solely
responsible for the observed disassembly. This is difficult to envision
given that
-SNAP was present in significant excess over its
membrane receptors in our experiments. The effects of NSF on fully
assembled SNARE complexes containing syntaxin, synaptobrevin, and
SNAP-25 are likely to be mediated by the same conformational change in
syntaxin, since syntaxin* also shows reduced ability to bind
synaptobrevin and SNAP-25 (Fig. 3B).
-SNAP binding (Fig. 5) has been shown previously to interact with
synaptobrevin, SNAP-25, the
1 subunit of the class B N-type
Ca
channel, and
synaptotagmin(27, 34, 49) .
This
domain contains a region of six heptad repeats which have a high
propensity to participate in
-helical coiled-coil protein-protein
interactions (see Ref. 34 for discussion). Such heptad repeats are also
found in the syntaxin-binding domains of SNAP-25 and synaptobrevin, and
it has been proposed that the protein-protein interactions between
members of the SNARE complex are mediated by assembly of coiled-coil
structures(27, 34, 46) .
-SNAP also
contains several regions with propensity to form coiled-coils (amino
acids 5-28, 127-150, and 166-188(6) ), and
there are hydrophobic forces involved in the binding between
-SNAP
and syntaxin as shown by its relative insensitivity to high salt
concentrations (Fig. 2B). We speculate that
-SNAP
also binds to the carboxyl-terminal domain of syntaxin via a
coiled-coil interaction. Synthetic peptides corresponding to two of
these predicted coiled coil regions have recently been reported to
interfere with
-SNAP function in exocytosis, implicating these
domains in functionally important protein-protein
interactions(51) .
-SNAP, the amino-terminal
part of the molecule appears to play an essential role in the
conformational change induced by NSF. This is documented by our finding
that the carboxyl-terminal fragment of syntaxin will support binding of
-SNAP and synaptobrevin but not their dissociation by NSF (Fig. 5C). Apparently, NSF induces an interaction
between the carboxyl- and amino-terminal regions of syntaxin which
results in loss of affinity for
-SNAP and other SNARE proteins. In
fact, it has previously been reported that amino- and carboxyl-terminal
fragments of syntaxin bind to each other and that this association
prevents the binding of synaptobrevin(27) . Since the
amino-terminal portion of syntaxin also contains domains with a high
propensity for the formation of coiled-coil interactions, it is
possible that the non-binding syntaxin* conformation is stabilized by
such interactions between amino- and carboxyl-terminal regions of the
molecule (see Fig. 6). NSF catalyzes, through
-SNAP, the
transition between two syntaxin conformations. NSF can therefore be
thought of as a member of the growing family of ``molecular
chaperone'' type proteins, which are important in regulating
protein conformation and the oligomerization of multi-subunit protein
complexes(52, 53) .
-SNAP and NSF poses the intriguing
question of whether in an intact neuron, where high concentrations of
-SNAP and NSF are present, they will bind to syntaxin that is not
part of an assembled SNARE complex. This is not a trivial consideration
since syntaxin is widely distributed in nonsynaptic areas of neuronal
plasma membranes with only a small portion likely to be in a SNARE
complex at any given time(19, 35) . Previous reports
have suggested that
-SNAP and NSF are both partially membrane
associated, and may therefore be interacting with membrane receptors
which are not part of a docking complex(47, 50) . It is
possible that the fully assembled SNARE complex has a higher affinity
for binding
-SNAP than the individual components do(48) .
However, our preliminary observations using purified recombinant
proteins suggest that affinity of
-SNAP for syntaxin is similar to
its affinity for the SNARE complex.
A further possibility
is that NSF effectively induces fusion only when it acts on an
appropriately assembled oligomeric SNAP receptor complex. It will be
interesting to study the subcellular localization of
-SNAP and NSF
in neurons more closely to see if they bind to membranes only at sites
where vesicles dock, or if they instead bind diffusely to membranes
with a distribution similar to that of syntaxin(19) .
S, adenosine
5`-O-(thiotriphosphate).
-NSF,
-SNAP, and
-SNAP, R.
H. Scheller and M. K. Bennett for cDNAs encoding syntaxin and
synaptobrevin, and T. C. Südhof for cDNA encoding SNAP-25. We are
grateful to Silke Engers for her outstanding work in generating the
hybridoma cell lines. We thank E. R. Chapman for helpful discussions
and several expression vector constructions, and J. M. Edwardson, D.
Bruns, members of the Jahn laboratory, and P. DeCamilli for helpful
discussions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.