From the Department of Physiology, University of Wisconsin, Madison, Wisconsin 53706
Received for publication, December 26, 2000, and in revised form, February 6, 2001
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ABSTRACT |
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Assembly of the plasma membrane proteins syntaxin
1A and SNAP-25 with the vesicle protein synaptobrevin
is a critical step in neuronal exocytosis. Syntaxin is anchored
to the inner face of presynaptic plasma membrane via a single
C-terminal membrane-spanning domain. Here we report that this
transmembrane domain plays a critical role in a wide range of syntaxin
protein-protein interactions. Truncations or deletions of the
membrane-spanning domain reduce synaptotagmin, Syntaxin 1A was initially identified as a 35 kDa protein in
the plasma membrane of amacrine cells (1), as a subunit of Ca2+ channels (2, 3) and as a synaptotagmin-binding protein (4). Since these initial reports, the function of syntaxin as a central
component in the synaptic vesicle membrane fusion machinery has been
well established (reviewed in Refs. 5-7). Syntaxin forms a putative
membrane fusion apparatus by assembling into a four-helix bundle (8)
with the plasma membrane protein SNAP-251 and the
synaptic vesicle protein synaptobrevin, to form a SNARE complex (9).
Assembly of this complex is necessary (10, 11) and may be sufficient to
drive membrane fusion (12-14). One current view is that the zippering
together of the four-helix bundle drives membrane fusion by pulling the
vesicle and target membranes together (8, 12, 15). In this model, the
transmembrane domains (TMDs) of synaptobrevin and syntaxin would form
part of a fusion pore (16). Thus, structure-function relationships of
these TMDs may reveal insights into the mechanism of membrane fusion
(13, 14, 17-19).
Syntaxin functions as a key element in membrane traffic and membrane
fusion by interacting with a wide range of other proteins. The many
binding partners of syntaxin, in excess of twenty, include rbSec1A/nsec-1/munc18 (20-22), CSP (23), syntaphilin (24), Biochemical studies of syntaxin, including structural determinations
(8, 33, 34), have made almost exclusive use of the cytoplasmic domain
of the protein. Yet, a number of reports indicate that the TMD of
syntaxin is a critical determinant for protein-protein interactions;
removal of the TMD inhibits synaptotagmin, synaptobrevin, and
To better understand syntaxin protein-protein interactions, we have
carried out a detailed investigation of the role of the syntaxin TMD in
mediating syntaxin-target protein interactions. We provide evidence
that the TMD of syntaxin affects the ability of its cytoplasmic domain
to engage partner proteins. The TMD fulfills this role in a
length- and sequence-specific manner that is not dependent upon
TMD-mediated dimerization. Furthermore, mutagenesis experiments
demonstrate that membrane insertion and wild-type partner protein
binding activity can be completely uncoupled. We propose that the TMD
affects target protein interactions by affecting the conformation of
the cytoplasmic domain of syntaxin.
Recombinant Proteins--
Mutagenesis (truncation, deletion, and
chimeric protein construction), expression, and purification of
recombinant proteins were carried out as described (38, 39). cDNA
to generate syx-mult and syx-A15 (19) were kindly provided by D. Langosch (Heidelberg, Germany). cDNA encoding rbSec1A (21, 40),
syntaxin 1A (4), synaptobrevin 2/VAMP2 (41), and synaptotagmin I (42)
were kindly provided by P. De Camilli (New Haven, CT), R. Scheller (Stanford, CA) and T. C. Sudhof (Dallas, TX).
Rat Brain Detergent Extracts--
Crude synaptosomes were
prepared by homogenization of 1-2 fresh rat brains in 320 mM sucrose buffer. The homogenate was centrifuged at 5000 rpm for 2 min in a Beckman JA-17 rotor; the pellet was discarded, and
the crude synaptosome fraction was collected by centrifuging the
supernatant at 11,000 rpm for 12 min in the same rotor. The resulting
pellet was resuspended in 25-30 ml of 50 mM HEPES, pH 7.4, 100 NaCl buffer plus 1% Triton X-100 and protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, and
20 µg/ml aprotinin) and solubilized for 30-45 min at 4 °C on a
rotator. Insoluble material was removed by centrifugation at 17,000 rpm
for 20 min in a Beckman JA-17 rotor. The final detergent extract
yielded 1 mg/ml protein, and 1-mg aliquots were incubated with 30 µg
of immobilized fusion protein as described below.
Binding Assays--
All binding assays were carried out by
immobilizing one protein on glutathione-Sepharose beads. Immobilized
fusion proteins were incubated with either purified soluble binding
partners in Tris-buffered saline (TBS; 20 mM Tris, 150 mM NaCl) plus 0.5% Triton X-100 or rat brain detergent
extracts (1 ml at 1 mg/ml, described above) with either 2 mM EGTA or 1 mM free Ca2+ for 1-2
h at 4 °C. Beads were washed three times in binding buffer. Bound
proteins were solubilized by boiling in SDS-sample buffer, subjected to
SDS-PAGE, and visualized by staining with Coomassie Blue or by
immunoblotting. For blotting, mouse monoclonal antibodies directed
against synaptotagmin I (604.4 and 41.1), In Vitro Transcription and Translation--
Wild-type and mutant
syntaxin cDNAs cloned into pGEX-2T were used as PCR templates using
a 5' primer containing the T7 promoter plus sequence complementary to
the 5'-end of pGEX-2T. The reverse primer was complementary to the
3'-end of pGEX-2T. PCR was carried out using 30 ng of plasmid DNA, 13.3 µM primers, and Pfu polymerase; samples were cycled 25 times (45 s at 95 °C, 45 s at 54 °C, and 2 min at 72 °C).
PCR products (0.5 µg) were then used directly in a TnT in
vitro transcription/translation system (Promega, Madison, WI) by
incubating with 25 µl of reaction mix containing reticulocyte lysate
and [35S]methionine according to the manufacturer's
instructions and with canine pancreatic microsomes added as indicated.
To determine incorporation of syntaxin into microsomal membranes, 5 µl of the translation mix was added to 400 µl of K-Glu buffer (120 mM potassium glutamate, 20 mM potassium
acetate, 2 mM EGTA, 20 mM HEPES, pH 7.2) and,
to pellet the membranes, centrifuged at 70,000 rpm for 30 min in a
Beckman TLA 100.3 rotor. As indicated, parallel samples were washed
with 400 µl of 100 mM Na2CO3
buffer, pH 11.5, and membranes were collected by centrifugation as
described above. Pellet and supernatant samples were solubilized by
boiling in reducing SDS-sample buffer, and equal fractions were
subjected to SDS-PAGE. Gels were processed for fluorography using
Amplify (Amersham Pharmacia Biotech) and fluorographs are shown in Fig. 7.
Previous studies indicated that removal of the transmembrane
domain (TMD) of syntaxin impaired synaptotagmin, synaptobrevin, and
/
-SNAP, and
synaptobrevin binding. In contrast, deletion of the transmembrane
domain potentiates SNAP-25 and rbSec1A/nsec-1/munc18 binding. Normal
partner protein binding activity of the isolated cytoplasmic domain
could be "rescued" by fusion to the transmembrane segments of
synaptobrevin and to a lesser extent, synaptotagmin. However,
efficient rescue was not achieved by replacing deleted transmembrane
segments with corresponding lengths of other hydrophobic amino acids.
Mutations reported to diminish the dimerization of the transmembrane
domain of syntaxin did not impair the interaction of full-length
syntaxin with other proteins. Finally, we observed that membrane
insertion and wild-type interactions with interacting proteins are not
correlated. We conclude that the transmembrane domain, via a
length-dependent and sequence-specific mechanism, affects
the ability of the cytoplasmic domain to engage other proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
-SNAP (9, 25), sec6/8 (26), tomosyn (27), Munc-13 (28), and as
mentioned above synaptotagmin (4, 29) as well as a growing assortment
of channels/receptors (see, for example Refs. 2, 3, 30-32).
/
-SNAP (25, 29) binding activity. In addition, insertion of the
transmembrane region into membranes is required for cleavage of
syntaxin by botulinum neurotoxin C1 (35, 36), and the membrane anchors
of syntaxin and synaptotobrevin are required for maximal stability of
the SNARE complex (37).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
-SNAP (77.1), SNAP-25
(71.2), and synaptobrevin II (69.1) were kindly provided by R. Jahn and
S. Engers (Gottingen, Germany). Immunoreactive bands were visualized
using enhanced chemiluminescence. Each binding assay was carried out in
at least three independent trials, and representative experiments are
shown in the figures.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
-SNAP binding activity (25, 29). Furthermore, syntaxin must be
anchored into lipid bilayers via its C-terminal membrane-spanning domain to be cleaved by botulinum neurotoxin C (35, 36). Finally, removal of the syntaxin TMD decreases the stability of fully assembled SNARE complexes (37). Whereas these reports suggested that the TMD of
syntaxin is important for protein-protein interactions, it was not
clear whether complete removal of the transmembrane segment of syntaxin
grossly affected the structure of the protein, or whether the TMD
played a more specific or direct role in mediating protein-protein
interactions. Therefore, we began to investigate the role of the
transmembrane domain in protein-protein interactions by constructing
more subtle truncations at the C terminus of the TMD. These constructs,
shown in Fig. 1A, were
expressed as GST fusion proteins and used as affinity matrices for the
binding of synaptotagmin I,
/
-SNAP, SNAP-25, and synaptobrevin II
present in Triton X-100 extracts of rat brain membranes. As shown in
Fig. 1B, truncation of the TMD resulted in the progressive
loss of synaptotagmin,
/
-SNAP, and synaptobrevin binding
activity. Even removal of the last two amino acids of the TMD slightly,
yet reproducibly, reduced synaptotagmin interactions. Truncation to
amino acid 281, which would be predicted to lie within the opposite
leaflet of the lipid bilayer relative to the cytoplasmic domain,
dramatically reduced binding. These data suggest that the distal region
of the TMD can affect the interaction of the H3 domain of syntaxin with
target proteins. We note that in these experiments, and in experiments
described below, similar results were observed using purified
recombinant proteins as the "ligands" in the GST pull-down assays
(data not shown). Thus, the interactions reported here are direct.
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Fig. 1.
Effect of C-terminal syntaxin truncations on
syntaxin protein-protein interactions. A, schematic of
syntaxin C-terminal TMD truncations. The Habc and H3 domains of
syntaxin are shaded, the TMD is shown in black.
The H3 and TMDs are enlarged to indicate the positions of the
C-terminal truncations. B, effect of syntaxin C-terminal TMD
truncations on protein-protein interactions. Thirty µg of GST or the
indicated versions of GST-syntaxin was immobilized on
glutathione-Sepharose beads. Beads were incubated with 1 mg of rat
brain detergent extract (1 mg/ml protein) for 2 h in Tris-buffered
saline with 1% Triton X-100 plus either 2 mM EGTA
( Ca2+) or 1 mM Ca2+
(+Ca2+). Beads were washed three times with binding buffer
and boiled in SDS sample buffer. Thirteen percent of the bound material
was subjected to SDS-PAGE, transferred to nitrocellulose, and probed
with primary antibodies directed against synaptotagmin I,
/
-SNAP,
SNAP-25, or synaptobrevin. Total corresponds to 5 µg of the rat brain
detergent extract. Binding of synaptotagmin,
/
-SNAP, and
synaptobrevin progressively decreases with increasing TMD truncations.
In contrast, SNAP-25 binding increases after truncation past residue
276. C and D, removal of the syntaxin
(syx) TMD increases rbSec1A binding activity. In
C, full-length syntaxin (1) and syntaxin lacking a TMD
(1) were used to affinity purify rbSec1A from rat brain detergent
extracts as described in B except that 20 mg of brain
extract were used for each sample, and samples were visualized by
staining with Coomassie Blue. In D, recombinant rbSec1A was
titrated onto either full-length syntaxin or syntaxin lacking a TMD;
again, bound rbSec1A was visualized by staining with Coomassie Blue. In
both experiments, rbSec1A binding was enhanced by removal of the
syntaxin TMD. The identity of native rbSec1 was confirmed by immunoblot
analysis (data not shown).
In contrast to the diminished binding of synaptotagmin, /
-SNAP,
and synaptobrevin, we observed that SNAP-25 binding was enhanced by
removal of the TMD, and this effect became apparent by truncating from
amino acid 276 back to residue 271 (Fig. 1B). These data
suggest that truncation of the TMD does not result in gross misfolding,
but rather can differentially affect the affinity of different
interacting proteins; this effect is consistent with a model in which
the TMD truncations can switch syntaxin between different
conformations, discussed further below. We also compared the
interactions of full-length and the cytoplasmic domain of syntaxin with
native (Fig. 1C) as well as recombinant
rbSec1A/nsec-1/munc18 (Fig. 1D). Analogous to SNAP-25,
rbSec1A bound more efficiently to the cytoplasmic domain of syntaxin
than to the full-length protein. Thus, removal of the TMD inhibits
synaptotagmin,
/
-SNAP, and synaptobrevin binding and enhances
SNAP-25 and rbSec1A binding.
To determine whether the effects of the syntaxin TMD truncations were
length- or position-sensitive, we shortened the TMD by internal
deletions at the N-terminal end of the TMD (shown schematically in Fig.
2A). As shown in Fig.
2B, removal of only two amino acid residues at the
N-terminal edge of the TMD reduced binding of synaptotagmin and
/
-SNAP. Binding of synaptotagmin was further reduced by
progressively larger deletions of four and seven amino acids.
Interestingly, synaptobrevin binding was largely unaffected by
N-terminal TMD deletions, indicating that these deletions did not
result in gross misfolding of syntaxin. Furthermore, SNAP-25 and
rbSec1A again showed an increase in binding, and this increase required
the removal of seven residues from the N-terminal side of the TMD. For
comparison, increased SNAP-25 binding was not observed until more than
twelve residues were removed from the C-terminal end of the syntaxin
TMD. These data suggest that the role of the TMD in syntaxin binding
interactions is complex and involves determinants other than simple
length requirements.
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We addressed this hypothesis via rescue experiments in which we tried
to restore wild-type binding interactions with the 1-281 truncation
and 265-270 deletion mutants by adding the appropriate number of
amino acids onto the C-terminal tail of the TMD. To test whether the
TMD must form an
-helix of a certain length, we added either a
string of isoleucines, which can form an
-helix, or a string of
alternating proline/phenylalanine residues (Pro/Phe), which cannot form
an
-helix (shown schematically in Fig.
3A). As shown in Fig.
3B, the 1-281 mutant showed diminished synaptotagmin,
/
-SNAP, and synaptobrevin binding activity. Again, SNAP-25
binding was not impaired, providing a positive control for the folding of the mutants. Interestingly, addition of seven Ile residues to the
end of the 281 mutant partially rescued
/
-SNAP and synaptobrevin binding, whereas the Pro/Phe sequence did not rescue binding of these
proteins. These data indicate that
/
-SNAP and synaptobrevin binding require a full-length TMD with the ability to form an
-helix. In contrast, neither the Ile nor the Pro/Phe sequences rescued synaptotagmin binding, again indicating that the TMD fulfills different requirements for the binding of different interacting proteins. Similar experiments were conducted using the
265-270 deletion mutant. Consistent with the data in Fig. 2, this deletion did
not affect synaptobrevin binding, but did affect the binding of all
other proteins examined (Fig. 3C). In this case, the Ile sequence failed to rescue synaptotagmin or
/
-SNAP binding
whereas, surprisingly, very low levels of rescue were observed with the non-helix forming Pro/Phe sequence. For these interactions, the ability
of added on residues to restore wild-type binding activity depended
upon whether the TMD was truncated at the N- or C-terminal end, as well
as on the content of the added-on sequence. In contrast, the enhanced
binding of SNAP-25 to the deletion mutant was partially abrogated by
both added-on sequence stretches. In summary, the data from these
rescue experiments suggest that the length, primary sequence,
and the position of the primary sequence of the TMD of syntaxin are all
key factors in syntaxin-target protein interactions.
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We further examined the sequence requirements of the syntaxin
TMD by constructing two chimeric syntaxins that harbored TMDs from
synaptobrevin II or synaptotagmin I (Fig.
4A). As shown in Fig.
4B, replacement of the syntaxin TMD with the synaptobrevin TMD resulted in a protein with wild-type, or stronger, synaptotagmin, /
-SNAP, and synaptobrevin binding activity. In contrast,
replacement with the synaptotagmin TMD resulted in only partial rescue
of synaptotagmin,
/
-SNAP, and synaptobrevin binding activity.
Because the TMDs of synaptobrevin and synaptotagmin are not homologous, these experiments demonstrate some degree of promiscuity in the sequence requirements within the TMD. These observations, coupled to
the findings that mutations on the distal side of the membrane anchor
(i.e. the N-terminal truncation mutants) can affect
protein-protein interactions (Fig. 1B), prompted us to
investigate the possibility that the role of the TMD is to induce
oligomerization of the H3 domain of syntaxin to drive normal
interactions with other proteins. Indeed, recent studies have
demonstrated that the transmembrane domain of syntaxin mediates
syntaxin homodimerization as well as binding to the TMD of
synaptobrevin (18, 19). The same may hold true of the TMD of
synaptotagmin, which has been recently shown to contain a novel
clustering site within the N-terminal half of the protein (43, 44). To
determine whether this oligomerization activity lies within the TMD of
synaptotagmin, we mapped the N-terminal clustering site. The constructs
used for this analysis (shown schematically in Fig.
5A) were immobilized as GST
fusion proteins and were assayed for their abilities to bind a
His6-tagged fragment of synaptotagmin (residues 1-265). As
shown in Fig. 5B, the soluble synaptotagmin fragment bound
to its immobilized counterpart in a Ca2+-independent
manner. Removal of the luminal domain did not inhibit binding, however
further truncation that removed the TMD strongly reduced binding
activity. These data indicate that the TMD of synaptotagmin could
directly mediate oligomerization of the protein, but it is also
possible that the TMD enables another region of synaptotagmin to
homo-oligomerize. We confirmed these results using native synaptotagmin
from brain detergent extracts as the ligand (Fig. 5C).
However, in these experiments, Ca2+ facilitated binding,
presumably because of Ca2+-triggered oligomerization of the
C2B domain of the protein (Refs. 39, 45-48).
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In summary, these results suggest that the TMDs of either synaptobrevin
or synaptotagmin may rescue deletion of the syntaxin TMD by conferring
oligomerization activity. To test this hypothesis directly, we made use
of a syntaxin TMD mutant, syx-mult, that harbors three amino acid
substitutions that block TMD-mediated oligomerization (Ref. 19; Fig.
6A). As a control, we also
analyzed a syntaxin mutant, syx-A15, which harbors a string of fifteen alanine residues from position 266-280 (Ref. 19; Fig. 6A).
The ability of these mutant syntaxins to bind partner proteins was tested as described in Fig. 1B. The syx-A15 binding profile
was indistinguishable from the minus TMD mutant, again demonstrating the sequence specificity of the TMDs to rescue wild-type binding activity. Surprisingly, syx-mult exhibited normal to enhanced synaptotagmin, /
-SNAP, and synaptobrevin binding activity. This result strongly indicates that the role of the TMD is not simply to
drive syntaxin into dimers for normal binding activity.
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Finally, we sought to determine whether the truncation, deletion, and
chimeric mutants were able to stably insert into membranes. We
postulated that impaired membrane insertion would be analogous to
impaired insertion into the detergent micelles used for our protein-protein interaction studies, resulting in alterations in the
disposition of the cytoplasmic domain along the surface of the membrane
or micelle (49). As shown in Fig.
7A, full-length syntaxin
(residues 1-288), associated with the pellet fraction in the presence
but not the absence of microsomal membranes. Furthermore, the
translated protein could not be removed from the microsomal membranes
upon extraction with pH 11 bicarbonate (the P2 fraction in Fig.
7A) but could be extracted with Triton X-100 (data not shown). These data demonstrate that syntaxin is properly inserted into
membranes in this in vitro transcription/translation system. We then tested the ability of the C-terminal truncation mutants to
stably insert into membranes. As shown in Fig. 7A, the 284, 281, and 276 truncation mutants inserted into membranes in a pH 11 extraction-resistant manner whereas the 271 mutant failed to bind
membranes. The ability of the 281 and 276 truncation mutants to stably
insert into membranes is notable, given that these mutations strongly
affect syntaxin protein-protein interactions. Clearly, membrane
insertion and target protein binding activity can be uncoupled. The
C-terminal deletion mutant 266-269 was also inserted into
membranes, but the larger
265-270 deletion mutant failed to become
incorporated into microsomes, either because of loss of targeting,
translocation, or stable insertion (Fig. 7B). It is notable
that the
265-270 deletion mutant exhibited wild-type synaptobrevin
II binding activity (Fig. 3C), further demonstrating that
membrane incorporation and partner protein binding activity can be
uncoupled. We also tested the membrane insertion activity of the
chimeric protein that displayed different interacting protein binding
avidities. The syntaxin-synaptotagmin-TMD and
syntaxin/synaptobrevin-TMD were incorporated into membranes with
similar efficiencies, despite their differential abilities to rescue
the loss of the syntaxin TMD. Thus, at the resolution of this assay
system, proper membrane insertion activity is not correlated with
wild-type syntaxin-partner protein binding activity. These data argue
against a model in which TMD mutations alter cytoplasmic domain
interaction by affecting the disposition of the protein relative to the
surface of the membrane or micelle. This argument is further supported
by the findings that removal of the TMD affects syntaxin
protein-protein interactions in other detergents, including detergents
with small aggregation numbers such that a surface (e.g.
CHAPS and cholate), to which the cytoplasmic domain could interact with
(49), is not formed (data not shown).
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In the final series of experiments we further established the
specificity of the syntaxin TMD in protein-protein interactions. These
experiments were carried out to rule out nonspecific direct binding
interactions between TMDs and the syntaxin interacting proteins
examined above. For this analysis we compared the TMDs of syntaxin and
synaptobrevin in greater detail. As shown in Fig. 8B, neither synaptotagmin nor
/
SNAP bound to full-length synaptobrevin, despite the presence
of the synaptobrevin TMD. As a positive control, the syntaxin construct
harboring the synaptobrevin TMD (also shown in Fig. 4) efficiently
bound both synaptotagmin or
/
SNAP. However, grafting the
syntaxin TMD onto the cytoplasmic domain of synaptobrevin did not
result in a chimeric protein with any synaptotagmin or
/
SNAP
binding activity. These data clearly demonstrate that the syntaxin TMD
is not sufficient to mediate binding of target proteins. Rather, some
TMDs enable conjoined cytoplasmic domains to bind other proteins with
higher or lower affinities. This effect may be true for other
interactions as well. For example, high affinity
SNAP-25·synaptobrevin interactions also require the TMD of
synaptobrevin (Fig. 8). Interestingly, high affinity SNAP-25 binding to
the cytoplasmic domain of synaptobrevin can be partially rescued by
grafting the syntaxin TMD onto synaptobrevin. Thus, the ability of TMDs
to affect cytoplasmic domain interactions with other proteins may be a
common phenomena among SNAREs.
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DISCUSSION |
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Previous studies indicated that the TMD of syntaxin plays a key
role in the ability of syntaxin to interact with other proteins; removal of the TMD inhibited synaptotagmin and /
SNAP-binding activity (25, 29). However, it was not clear from these studies whether
complete deletion of the TMD had gross effects on the folding of
syntaxin, whether the TMD directly participated in target protein
binding interactions such that its deletion inhibited binding (18, 19),
or whether the TMD contributed to protein-protein interactions via a
trivial nonspecific sticky effect. Here we have addressed each of these
issues and provide data to indicate that syntaxin can exist in
different conformations that are influenced by its TMD.
We began by showing that subtle deletions/truncations of the syntaxin
TMD inhibited binding of synaptotagmin, /
SNAP, and synaptobrevin in a graded manner. Strikingly, removal of only two
residues at the C terminus of the TMD reproducibly inhibited synaptotagmin binding. It is possible that the loss of synaptotagmin binding activity could be caused by loss of direct interactions between
the TMDs of these proteins (Fig. 1B). For example, it has
been reported that the TMD of syntaxin interacts directly with the TMD
of synaptobrevin (18, 19). However, this does not appear to be the
case. For example, grafting the syntaxin TMD onto the cytoplasmic
domain of synaptobrevin did not result in a protein that bound to
either synaptotagmin or
/
SNAP (Fig. 8). In addition, we have
observed that removal of the syntaxin TMD largely abolishes the
co-immunoprecipitation of syntaxin with the purified cytoplasmic domain
of synaptotagmin (data not shown). Clearly, neither the cytoplasmic
domain of synaptotagmin, nor
/
SNAP, have access to the outer
leaflet of the plasma membrane that contains the distal region of the
syntaxin TMD or to the inside of a detergent micelle as in our
experimental conditions, so it is unlikely that the TMD of syntaxin
plays a direct role in binding these proteins. Rather, these data
indicate that the TMD of syntaxin somehow influences the ability of its
cytoplasmic domain, and in particular the H3 domain to bind
synaptotagmin (29, 50, 51) and
/
SNAP (25, 52, 53).
As noted above, the TMD of synaptobrevin has been reported to interact
directly with the TMD of syntaxin (18, 19). In this case, removal of
the TMD of syntaxin would be expected to inhibit binding of
synaptobrevin. However, this does not appear to be the reason why
removal of syntaxin TMD segments inhibits synaptobrevin binding in our
experiments (Figs. 1B and 2B). This conclusion
stems from our use of point mutations that inhibit the interaction
between the TMD of syntaxin and synaptobrevin (19). As shown in Fig.
6B, these loss-of-function mutants actually facilitated
association of these proteins under our assay conditions (Fig.
6B). We conclude that the TMD of syntaxin indirectly
influences the ability of syntaxin to bind not only synaptotagmin and
/
SNAP, but also synaptobrevin. Whereas C-terminal truncations
had graded effects on the binding of all three of these proteins,
N-terminal deletions did not diminish synaptobrevin binding but did
inhibit synaptotagmin and
/
SNAP binding (e.g.
265-270; Fig. 2B). These findings demonstrate that there
are distinct requirements within the syntaxin TMD that are important
for the binding of different proteins. Because synaptotagmin and
/
SNAP can bind and partially penetrate into membranes (43, 51,
54, 55), it is possible that their binding sites extend a short
distance into the plane of the bilayer, lending increased sensitivity
to TMD N-terminal deletions.
In contrast to the diminished binding of synaptotagmin, synaptobrevin,
and /
SNAP upon removal of portions of the syntaxin TMD, binding
of SNAP-25 and rbSec1A is enhanced. These data argue against simple
misfolding of the syntaxin TMD mutants. Because neither SNAP-25 nor
rbSec1A have direct access to the TMD of syntaxin, these observations
provide further support for a model in which the TMD affects
cytoplasmic domain protein-protein interactions by influencing the
conformation of the H3 domain.
It is critical to note the effects of the syntaxin TMD on
protein-protein interactions is sequence specific and is not because of
nonspecific binding of a hydrophobic TMD to other proteins. As shown in
Figs. 3, 4, 6, and 8, a variety of full-length hydrophobic TMDs,
grafted onto either syntaxin or onto synaptobrevin, fail to bind
synaptotagmin, /
SNAP, and synaptobrevin. Furthermore, the
265-270 deletion inhibits binding of synaptotagmin and
/
SNAP, but does not inhibit synaptobrevin binding. Thus,
shortening the TMD does not simply allow proteins to "fall off."
Our results demonstrate that the cytoplasmic domain of syntaxin must be
connected to the proper length and sequence TMD to exhibit the correct
binding interaction profile.
We suggest two possibilities for how the TMD of syntaxin can
influence the interaction of its cytoplasmic domain with other proteins, either positively (synaptotagmin, synaptobrevin,
/
SNAP) or negatively (rbSec1A, SNAP-25). In one model,
homo-oligomerization of the TMD (19) results in multimerization of the
cytoplasmic domain of the protein, and this oligomerization facilitates
some interactions (synaptotagmin, synaptobrevin, and
/
-SNAP) and inhibits others (SNAP-25 and rbSec1A). In this model, removal of the
TMD would inhibit binding of the former set of proteins and facilitate
binding of the latter set. This model was prompted by recent reports
establishing that the TMD of syntaxin directly mediates oligomerization
of the protein (19). We tested this model by examining the ability of a
mutant syntaxin, that fails to oligomerize via the TMD, to bind other
proteins. We found that these mutations did not inhibit synaptotagmin,
synaptobrevin or
/
SNAP binding and did not enhance SNAP-25 or
rbSec1A binding (Fig. 6B). Whereas we cannot rule out the
possibility that the syntaxin oligomerization mutant exhibits some
residual oligomerization activity, these data strongly argue that
the effects of TMD mutations on syntaxin interactions are not simply
secondary to effects on syntaxin self-association activity.
In the second model, syntaxin exists in multiple conformations that can
be influenced by the presence or absence of the TMD. In this model,
removal of the TMD favors a conformation in which the H3 domain has
impaired interactions with synaptotagmin, synaptobrevin, and
/
SNAP and has more favorable interactions with SNAP-25 and
rbSec1A. Indeed, syntaxin has been shown to exist in at least two
states; an open and a closed conformation (8, 33, 56). In the closed
conformation, part of the H3 domain forms a four-helix bundle with the
N-terminal Habc domain (33). Because the closed conformation forms a
high affinity complex with rbSec1A (34), it is tempting to speculate
that removal of the TMD favors this conformation, whereas the
full-length protein favors the open conformation. This model would also
account for the ability of the full-length open conformation to bind
efficiently to synaptobrevin, synaptotagmin, and
/
SNAP, because
all three proteins bind directly to the H3 domain that is exposed in
this conformation (33, 56). However, this two-conformation model does
not account for our SNAP-25 binding data, because SNAP-25 also
assembles onto the exposed H3 domain (57). This finding suggests that
syntaxin can adopt additional conformations, and the presence or
absence of a TMD favors one conformation over another to influence the affinity of target protein interactions.
In binary complexes with SNAP-25 or rbSec1A, the C-terminal region of
the cytoplasmic domain of syntaxin is disordered and does not form
direct contacts with these interacting proteins (33, 34). This region
becomes more ordered only upon assembly into SNARE complexes (8).
However, the studies indicating that there is a discontinuity in the
ordering of the structure between the TMD and the H3 domain, made use
of the cytoplasmic domain of syntaxin. It is possible that this segment
becomes ordered upon inclusion of the TMD and is involved in
transmitting structural information from the TMD to the H3 domain in
the context of the full-length protein. Understanding how the TMD
affects the structure of the cytoplasmic domain will require high
resolution structural studies focused on full-length syntaxin. In
summary, we propose that the TMD can inhibit binding of SNAP-25 and
rbSec1A, and can facilitate binding of synaptotagmin, /
SNAP,
and synaptobrevin by influencing the structure of the cytoplasmic
domain of the protein.
Our studies demonstrate that the length and sequence of the syntaxin
TMD are critical determinants for the specific interaction of syntaxin
with other proteins. In this light it is interesting to note that new
isoforms and splice variants of syntaxin have been reported that lack
the C-terminal transmembrane domain (58, 59). In some cases, truncated
forms of syntaxin protein have been detected and shown to exhibit
differential interactions with target proteins (59). Thus, removal of
the TMD via alternative splicing may strongly influence the function of
syntaxin in different cell types or different trafficking pathways.
Furthermore, reconstitution experiments indicate that SNARE-catalyzed
membrane fusion requires that only one v-SNARE and one t-SNARE need to
have transmembrane anchors; the other two strands of the SNARE complex
four-helix bundle do not require membrane anchors (13, 14, 60). These findings suggest that in some trafficking pathways, the SNARE complex
could contain syntaxins that are not anchored to the membrane via a TMD
as long as they are paired with another t-SNARE that has a
transmembrane domain. Thus, splicing TMDs in and of t-SNAREs may result
in SNARE complexes with unique properties.
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ACKNOWLEDGEMENTS |
---|
We thank Reinhard Jahn and Silke Engers for monoclonal antibodies, Tom Martin for the anti-synaptotagmin C2B domain antibody, Dieter Langosch for the syx-mult and syx-A15 cDNA, Tom Sudhof for synaptotagmin Ia cDNA, Richard Scheller for syntaxin IA and synaptobrevin/VAMP II cDNA, Pietro De Camilli for rbSec1A cDNA, and Jihong Bai and Mark Krebs for helpful discussions.
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FOOTNOTES |
---|
* This study was supported by Grant GM 56827-01 from the National Institutes of Health, Grant 9750326N from the American Heart Association, and the Milwaukee Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
A Pew Scholar in the Biomedical Sciences. To whom correspondence
should be addressed: Dept. of Physiology, SMI 129, University of
Wisconsin, 1300 University Ave., Madison, WI 53706. Tel.: 608-263-1762; Fax: (608) 265-5512; E-mail: chapman@physiology.wisc.edu.
Published, JBC Papers in Press, February 9, 2001, DOI 10.1074/jbc.M011687200
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ABBREVIATIONS |
---|
The abbreviations used are:
SNAP-25, synaptosome-associated protein of 25 kDa;
/
-SNAP, soluble NSF
attachment protein;
SNARE, soluble NSF attachment protein receptor;
TMD, transmembrane domain;
PAGE, polyacrylamide gel electrophoresis;
PCR, polymerase chain reaction;
GST, glutathione
S-transferase;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
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