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J Biol Chem, Vol. 275, Issue 4, 2959-2965, January 28, 2000
From the Experimental Immunology Branch, NCI, National Institutes
of Health, Bethesda, Maryland 20892
The docking and fusion of synaptic vesicles with
the presynaptic plasma membrane require the interaction of the
vesicle-associated membrane protein VAMP with the plasma membrane
proteins syntaxin and SNAP-25. Both of these proteins behave as
integral membrane proteins, although they are unusual in that they
insert into membranes post-translationally. Whereas VAMP and syntaxin
possess hydrophobic transmembrane domains, SNAP-25 does not, and it is
widely believed that SNAP-25 traffics to and inserts into membranes by
post-translational palmitoylation. In pulse-chase biosynthesis studies,
we now show that SNAP-25 and syntaxin rapidly bind to each other while
still in the cytosol of neuroendocrine and transfected heterologous cells. Cell fractionation studies revealed that cytosolic
SNAP-25·syntaxin complexes then traffic to and insert into
membranes. Furthermore, the association of SNAP-25 with membranes
is dramatically enhanced by syntaxin, and the transmembrane domain of
syntaxin is essential for this effect. Surprisingly, despite the
importance of the SNAP-25 palmitoylation domain for membrane anchoring
at steady state, removal of this domain did not inhibit the initial
association of newly synthesized SNAP-25 with membranes in the presence
of syntaxin. These data demonstrate that the initial attachment of newly synthesized SNAP-25 to membranes is a consequence of its association with syntaxin and that it is only after syntaxin-mediated membrane tethering that SNAP-25 is palmitoylated.
Calcium-stimulated exocytosis of synaptic vesicles is essential
for the development of the central nervous system as well as the
maintenance of proper neural signaling. For this reason, investigation
of the proteins regulating synaptic vesicle docking and fusion with
presynaptic plasma membranes is essential for a complete understanding
of the mechanisms regulating this complex process. Through a
combination of genetic and biochemical approaches, members of the
SNARE family of proteins have been
strongly implicated in the processes of vesicle docking and fusion
(reviewed in Refs. 1-3). On the synaptic vesicle itself is a member of
the vesicle SNARE family of VAMP-like proteins, whereas on the
presynaptic plasma membrane, there are
the target SNARE (t-SNARE)1 proteins syntaxin and SNAP-25.
To form a functional t-SNARE complex, syntaxin must first associate
with SNAP-25 (4). The binding of syntaxin to SNAP-25 induces a
conformational change in SNAP-25 (5), and it has been demonstrated that
a preformed t-SNARE complex is necessary for the subsequent formation
of the complete vesicle SNARE·t-SNARE complex (6). Following SNARE
complex assembly, a poorly understood series of protein-protein,
protein-lipid, and lipid-lipid interactions occur, with the net result
of all of these interactions being the fusion of the opposing membranes and the release of neurotransmitter into the synaptic cleft.
The t-SNARE syntaxin and the vesicle SNARE VAMP are carboxyl
terminus-anchored transmembrane proteins with typical hydrophobic transmembrane domains. However, these proteins are unusual in that
unlike most transmembrane proteins, they lack the signal sequence
required for cotranslational insertion into the endoplasmic reticulum
membrane and associate with membranes post-translationally (7, 8).
Although the t-SNARE SNAP-25 does not possess a hydrophobic
transmembrane domain like syntaxin or VAMP, it does possess a central
cysteine-rich "palmitoylation domain," and the association of
SNAP-25 with cellular membranes has been largely attributed to
acylation of the molecule (9-13). Indeed, deletion of the entire
palmitoylation domain (10) or mutagenesis of individual cysteine
palmitate acceptor sites (11) almost completely prevents membrane
association of mutant SNAP-25 at steady state.
Syntaxin, VAMP, and SNAP-25 each post-translationally insert into
membranes in the early secretory pathway, most likely the endoplasmic
reticulum. From there, these molecules are believed to traffic through
the Golgi to their site of action. At least for VAMP, insertion in
proteoliposome membranes is ATP-dependent, but does not
require the signal recognition particle receptor or the Sec61p complex
(8), highlighting the novel mechanism of membrane insertion of this
class of proteins. The trafficking of SNAP-25 to the plasma membrane of
PC12 cells also follows the secretory pathway, as treatment of these
cells with brefeldin A prevents plasma membrane targeting of SNAP-25
(12). Most important, this treatment almost completely prevents
palmitoylation of SNAP-25, strongly suggesting that SNAP-25 must
traffic to the plasma membrane before it can be palmitoylated
(12).
Although it is clear that palmitoylation alone can immobilize proteins
onto membranes, it is likely that palmitoylation substrates must first
associate (either transiently or stably) with membranes by a mechanism
independent of palmitoylation (14). For example, members of the Src
family of tyrosine kinases are cotranslationally myristoylated, and
this modification is essential for rapid membrane association and
palmitoylation of the Src family member p59fyn (15). Similarly,
palmitoylation and membrane association of the G protein
Unlike Src or G protein family members, SNAP-25 is not myristoylated,
leaving unresolved the mechanism by which SNAP-25 initially associates
with membranes, traffics through the secretory pathway, and is
expressed on the plasma membrane, where it is subsequently palmitoylated. Since syntaxin efficiently inserts into membranes even
in the absence of SNAP-25 (20), we investigated the biosynthesis and
membrane association of the SNAP-25·syntaxin t-SNARE complex in the
rat neuroendocrine PC12 cell line and in transfected heterologous cells. Using pulse-chase biosynthesis studies, we now show that newly
synthesized SNAP-25 associates with syntaxin almost immediately after
translation while the molecule still resides in the cytosol. Furthermore, we demonstrate that the initial association of SNAP-25 with membranes does not require palmitoylation, as a palmitoylation mutant of SNAP-25 associates with cell membranes as efficiently as does
wild-type (palmitoylated) SNAP-25. Rather, it is the binding of SNAP-25
to syntaxin that initially anchors SNAP-25 to membranes, and the
hydrophobic transmembrane domain of the syntaxin molecule is essential
for membrane localization of the SNAP-25·syntaxin heterodimer.
Materials--
Human SNAP-25b in the mammalian expression vector
pcDNA3 has been described previously (21). The cDNA encoding
rat syntaxin 1A was obtained from Dr. Richard Scheller (Stanford
University, Stanford, CA) and was subcloned into the
HindIII/XbaI sites of pcDNA3. The
palmitoylation mutant of SNAP-25 (SNAP-25 Cell Culture and Transfections--
The rat pheochromocytoma
cell line PC12 (American Type Culture Collection, Manassas, VA) was
maintained in Dulbecco's modified Eagle's medium containing 5% horse
serum and 10% fetal calf serum, and the human cervical carcinoma cell
line HeLa was maintained in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum in 75-cm2 tissue
culture flasks. Subconfluent HeLa cells were transiently transfected
with the indicated cDNAs using LipofectAMINE Plus reagent (Life
Technologies, Inc.). The cells were metabolically labeled 18-24 h
after transfection. Mock transfections were performed using the empty
pcDNA3 expression vector.
Metabolic Labeling and Immunoprecipitation--
PC12 cells and
transiently transfected HeLa cells, each cultured in 10-cm tissue
culture dishes, were washed with methionine-deficient Dulbecco's
modified Eagle's medium and pulse-labeled with 0.25 mCi of
[35S]methionine (ICN, Costa Mesa, CA) for either 15 min
or 1 h and chased for up to 3 h (as indicated in the figure
legends) at 37 °C. Cells were lysed in 1% Triton X-100 in
Tris-buffered saline on ice; nuclei and other debris were removed by
centrifugation; and specific immunoprecipitations using antibodies
bound to protein A-Sepharose were performed as described (22).
Immunoprecipitated proteins were analyzed on 10.5% SDS-polyacrylamide
gels; either the proteins were then immunoblotted using enhanced
chemiluminescence (ECL, NEN Life Science Products), or the gels were
impregnated with Enlightening (NEN Life Science Products) and analyzed
by fluorography. Band intensities from metabolic radiolabeling studies were quantitated using a Molecular Dynamics PhosphorImager, whereas those of immunoblots were quantitated using a Molecular Dynamics densitometer. X-ray films were digitally scanned, and composite figures
were generated using Adobe Photoshop.
Subcellular Fractionation--
Adherent cells were harvested by
trypsinization, and ~1 × 107 cells were resuspended
in 0.5 ml of hypotonic buffer (10 mM Tris, 10 mM KCl, 1 mM EGTA, and 0.5 mM
MgCl2, pH 7.4) and homogenized using a ball-bearing
homogenizer (clearance of 10 µm) at 4 °C. The homogenate was
centrifuged at 2000 × g for 5 min at 4 °C to obtain
a post-nuclear supernatant. This post-nuclear supernatant was
centrifuged at 110,000 × g for 20 min at 4 °C. The
supernatant (cytosolic) and pellet (membrane) fractions were then
brought to equal volumes in a final buffer containing a 1:1 mixture of hypotonic buffer and Triton lysis buffer. Immunoprecipitations using
the specific antibodies described above were performed on equivalent
fractions of each pool, and the immunoprecipitates were analyzed by
SDS-PAGE and fluorography or by immunoblotting.
SNAP-25 and Syntaxin 1 Associate in the Cytosol following
Biosynthesis in PC12 Cells--
At steady state, both SNAP-25 and
syntaxin 1 are associated almost exclusively with neuronal membranes
(23, 24). In addition, SNAP-25·syntaxin t-SNARE complexes have been
detected not only at the presynaptic membrane, but also in transport
vesicles undergoing fast axonal transport (25, 26), demonstrating that
t-SNARE complex assembly can occur prior to the arrival of these
proteins at the plasma membrane. Since each of these proteins
associates with membranes post-translationally, we examined the
kinetics of t-SNARE assembly in vivo to determine if the
components of the t-SNARE heterodimer come to reside on cellular
membranes independently or as pre-assembled complexes.
Pulse radiolabeling of the rat neuroendocrine PC12 cell line with
[35S]methionine followed by cell lysis and
immunoprecipitation with t-SNARE-specific antibodies revealed that
syntaxin associated with SNAP-25 within 15 min of synthesis of the
35S-labeled SNAP-25 polypeptide (Fig.
1A). Interestingly, we found that the amount of complex assembly observed after the pulse labeling was similar to that observed after a 3-h chase period in complete medium, demonstrating that t-SNARE complex assembly is rapid in PC12
cells. Sequential immunoprecipitation studies confirmed that each
antibody removed the majority of its ligand from the cell extract, and
quantitative analysis of the immunoprecipitates revealed that ~20%
of the pool of newly synthesized SNAP-25 was bound to syntaxin (data
not shown). In addition, SNAP-25·syntaxin t-SNARE complexes were not
observed when heterologous cells expressing SNAP-25 alone and syntaxin
alone were mixed prior to lysis and immunoprecipitation (data not
shown), indicating that the t-SNARE complexes observed above were not a
post-solubilization artifact.
Both syntaxin and SNAP-25 begin to associate with membranes ~20 min
after the completion of protein synthesis (12, 20). Since t-SNARE
complex formation was essentially complete after our 15-min pulse
labeling, we investigated the possibility that the t-SNARE complex
resided in the cytosol prior to the insertion of the complex into
membranes. PC12 cells were metabolically labeled with
[35S]methionine for 1 h, and isolated subcellular
fractions were analyzed by immunoprecipitation using control,
anti-syntaxin, or anti-SNAP-25 antibodies. In agreement with previous
studies, the anti-SNAP-25 immunoprecipitate revealed the presence of
newly synthesized SNAP-25 in both the cytosolic and membrane fractions of these cells (Fig. 1B). In addition, the anti-syntaxin
immunoprecipitate revealed that t-SNARE complexes containing syntaxin
and 35S-labeled SNAP-25 were present in both the cytosolic
and membrane fraction of these cells after pulse labeling (Fig.
1B), clearly demonstrating that newly synthesized t-SNARE
complexes are present in the cytosol of PC12 cells.
Quantitative immunoblotting confirmed that intracellular membranes did
not contaminate our cytosolic preparations. The isolated cytosolic
fraction of PC12 cells contained barely detectable levels of the
integral membrane protein calnexin, undetectable levels of syntaxin,
and 12% of the total pool of SNAP-25 (Fig. 1C). In addition, the cytosolic fraction did not contain detectable levels of
the vesicle protein VAMP (Fig. 1C). Taken together, these
results strongly argue against the possibility that endoplasmic
reticulum membranes, plasma membranes, or synaptic-like microvesicle
membranes contaminated our cytosol. The small amount of SNAP-25 present in the cytosol of PC12 cells is in agreement with previous results and
represents the non-palmitoylated pool of SNAP-25 (10, 12). Taken as a
whole, these data confirm that our isolated cytosol was not
significantly contaminated with membranes.
Syntaxin Enhances SNAP-25 Association with Membranes--
To more
precisely address the mechanism by which SNAP-25 and syntaxin associate
with membranes in neuronal cells in vivo, we adopted a
transfection system in heterologous cells using a variety of wild-type
and mutant t-SNARE proteins. The HeLa cell line chosen for these
studies does not express endogenous SNAP-25 or syntaxin 1 (data not
shown) and therefore afforded us a means to assess the role of each of
these proteins in membrane anchoring of t-SNARE complexes. We have
previously demonstrated efficient palmitoylation of SNAP-25 in these
cells under our experimental conditions, and the degree of
palmitoylation of SNAP-25 correlated with the extent of membrane
binding (27). Pulse-chase biosynthesis studies in transfected HeLa
cells revealed that when expressed alone, 35S-labeled
SNAP-25 was originally present in the cytosol, and even after a 3-h
chase, only a small fraction of the pool of 35S-labeled
SNAP-25 was associated with membranes (Fig.
2A). In agreement with the
results obtained in PC12 cells, coexpression with syntaxin resulted in
the detection of complexes of 35S-labeled SNAP-25 with
35S-labeled syntaxin in the cytosolic fraction from
pulse-labeled cells (Fig. 2B). In addition, coexpression
with syntaxin dramatically increased the association of
35S-labeled SNAP-25 with the membrane fraction after a 3-h
chase, and much of the 35S-labeled SNAP-25 present in the
membranes was associated with 35S-labeled syntaxin (Fig.
2B), demonstrating that SNAP-25·syntaxin t-SNARE complexes
formed in the cytosol trafficked to the membrane fraction during the
chase period.
During the course of these studies, we routinely observed that
coexpression with syntaxin protected SNAP-25 from degradation. Whereas
35S-labeled SNAP-25, when expressed alone, was rapidly
degraded during a 3-h chase (Fig. 2A), under identical
culture conditions, there was essentially a complete recovery of
35S-labeled SNAP-25 in cells expressing syntaxin (Fig.
2B). This argues that binding to syntaxin, a process known
to alter the conformation of SNAP-25 (5), also stabilizes SNAP-25 from
(presumably cytosolic) degradation.
To confirm the integrity of our cytosolic and membrane preparations in
our transfection system, HeLa cells transiently expressing Tac, the
interleukin-2 receptor
We performed quantitative PhosphorImager analysis to assess the
contribution of syntaxin to the membrane association of SNAP-25. Whereas only 14% of the total pool of 35S-labeled SNAP-25
was membrane-associated after a 3-h chase in the absence of syntaxin,
coexpression with syntaxin resulted in the association of 47% of
35S-labeled SNAP-25 with membranes (Fig.
3). Furthermore, under these conditions,
a sizable fraction (22%) of 35S-labeled SNAP-25 was
membrane-associated even after the short pulse labeling, clearly
showing that coexpression of syntaxin significantly enhances the
association of newly synthesized SNAP-25 with membranes.
The Palmitoylation Domain of SNAP-25 Is Insufficient for Membrane
Association of the t-SNARE Complex--
Since syntaxin rapidly binds
to SNAP-25 and also significantly enhances the association of SNAP-25
with membranes, we investigated the possibility that the syntaxin
transmembrane domain contributed to the enhanced binding of SNAP-25 to
membranes. We therefore generated a syntaxin mutant lacking the
carboxyl-terminal transmembrane domain, and pulse-chase biosynthesis
studies confirmed that whereas wild-type syntaxin resided exclusively
in the membrane fraction, the transmembrane mutant of syntaxin
(syntaxin
When coexpressed, wild-type syntaxin and wild-type SNAP-25 formed
complexes that were present in both the cytosolic and membrane fractions (Fig. 5A). Since
SNAP-25 was synthesized in excess of syntaxin in these studies, the
anti-syntaxin immunoprecipitate indicates the status of the
SNAP-25·syntaxin t-SNARE complex, and analysis of this
immunoprecipitate revealed that most t-SNARE complexes were present in
the membrane after the 3-h chase (Fig. 5A). Coexpression of
syntaxin
Since SNAP-25 association with membranes has been attributed to fatty
acylation in the central palmitoylation domain of SNAP-25 (9-13), we
assumed that wild-type SNAP-25 would bring syntaxin SNAP-25 Binding to Membranes Is Regulated by the Transmembrane
Domain of Syntaxin--
Since the palmitoylation domain of SNAP-25 is
insufficient to tether SNAP-25 to membranes in the presence of syntaxin
It is widely believed that a preformed t-SNARE complex composed of
SNAP-25 with syntaxin is essential for synaptic vesicles to dock and
ultimately fuse with the presynaptic plasma membrane. Many
investigators have used the pheochromocytoma PC12 cell line to
investigate the mechanisms responsible for the association of neuronal
SNARE proteins with membranes (7, 8, 10-12, 28). However, to our
knowledge, the biosynthesis of SNAP-25·syntaxin complexes in
vivo has not been addressed in this or any other cell type. We
have recently found that syntaxin and SNAP-25 bind to each other very
efficiently in transfected heterologous cells (27), and we have now
examined the assembly and membrane association of newly synthesized
endogenous t-SNARE complexes in PC12 cells. Using pulse-chase
biosynthesis studies, we have found that SNAP-25 and the transmembrane
t-SNARE syntaxin rapidly associate in these cells. In addition,
subcellular fractionation studies confirmed that a significant pool of
these t-SNARE complexes resides in the cytosol after a short pulse
radiolabeling and traffics to membranes during a 3-h chase period,
demonstrating that the t-SNARE complex of syntaxin and SNAP-25 forms in
the cytosol prior to its translocation to membranes.
To more completely study the mechanism of t-SNARE assembly, pulse-chase
metabolic labeling studies in transfected heterologous cells were used
to follow the trafficking of newly synthesized t-SNARE proteins to
membranes. In addition to confirming our PC12 data revealing the
presence of newly synthesized t-SNARE complexes in the cytosol, this
approach revealed that the association of SNAP-25 with membranes was
dramatically enhanced when SNAP-25 was coexpressed with wild-type
syntaxin. Surprisingly, membrane association of wild-type SNAP-25 was
almost completely blocked when SNAP-25 was coexpressed with a cytosolic
transmembrane mutant of syntaxin, demonstrating that the transmembrane
domain of syntaxin is responsible for the initial association of
SNAP-25 with membranes.
As further evidence for the importance of the transmembrane domain of
syntaxin in SNAP-25 targeting, we found that even a palmitoylation
mutant of SNAP-25 associated with membranes when coexpressed with
wild-type syntaxin, and in this case, the extent of initial membrane
association was comparable to that of wild-type SNAP-25. It is
important to emphasize that, in this work, we have specifically
addressed the mechanism of membrane targeting of newly
synthesized SNAP-25 and not the palmitoylation of membrane-bound SNAP-25, an event that occurs later in the lifetime of the molecule. Numerous studies have demonstrated that palmitoylation of SNAP-25 is
required for its stable association with membranes at steady state and
that SNAP-25 palmitoylation mutants reside in the cytosol (9-13). We
have obtained similar results by Western blot analysis of wild-type and
mutant SNAP-25 (data not shown). However, in this study, we have
concerned ourselves with the initial interaction of newly synthesized
SNAP-25 with membranes, and our data clearly show that membrane
association of either wild-type SNAP-25 or a palmitoylation mutant of
SNAP-25 is dramatically enhanced by its binding to newly synthesized syntaxin.
One of the most surprising findings of this study was our isolation of
newly synthesized SNAP-25·syntaxin complexes in the cytosol of PC12
cells or transfected heterologous cells. It is highly unlikely that
this result is a consequence of membrane contamination of our cytosol,
as the cytosolic fraction contained undetectable levels of the synaptic
vesicle membrane protein VAMP and only ~6% of the total pool of
syntaxin. This is important since ~15% of all SNAP-25 and syntaxin
are present on synaptic vesicles (29), and synaptic-like microvesicle
contamination of our cytosol would significantly compromise our
results. Like many C-terminal anchored membrane proteins, syntaxin,
VAMP, and SNAP-25 are synthesized on cytosolic ribosomes and associate
with membranes only after the completion of protein synthesis using a
novel "post-translational" mechanism of membrane insertion (7, 8).
Therefore, it is not surprising to find a small pool of newly
translated protein in the cytosol en route to membranes. Since the
association of newly synthesized syntaxin with membranes is rapid (20)
and membrane-associated syntaxin is quite stable, the vast majority of
syntaxin resides in the membrane fraction at steady state. Despite the
fact that the kinetics of SNAP-25 trafficking to membranes are
relatively slow, we and others have found that the half-life of the
total pool of SNAP-25 in PC12 cells is ~8 h
(11) and that the half-life of
syntaxin-associated (and presumably palmitoylated) SNAP-25 in these
same cells is ~15 h.2 Therefore, like syntaxin, SNAP-25
is also relatively stable, which favors the distribution of SNAP-25
into the membrane fraction at steady state.
Newly synthesized SNAP-25 inserts into membranes only poorly in the
absence of wild-type syntaxin or in the presence of a syntaxin
transmembrane mutant; and thus, syntaxin appears to be functioning as a
true molecular chaperone, escorting SNAP-25 into the secretory pathway,
where it will ultimately be palmitoylated. This also suggests that the
palmitoylation domain alone does not efficiently target SNAP-25 to
membranes. In agreement with this, Gonzalo et al. (13)
recently showed that a SNAP-25/green fluorescent protein chimera
containing the central region of SNAP-25 could traffic to membranes.
However, mutagenesis of Gln116, Pro117, and
Arg119, which reside outside of the palmitoylation domain,
prevented membrane attachment and palmitoylation, leading the authors
to propose that this mutation prevented protein-protein interactions necessary for membrane tethering of SNAP-25 (13). Since the chimera
used in their study presumably trafficked to membranes independently of
syntaxin, it is likely that other molecular chaperones can usurp the
role of syntaxin in escorting SNAP-25 to the secretory pathway. Thus,
even their study supports our hypothesis that the mere presence of the
palmitoylation domain does not guarantee that SNAP-25 will efficiently
insert into membranes.
Our finding that the palmitoylation domain of SNAP-25 is not required
for its initial association with membranes is indeed novel. Clearly,
palmitoylation is required for the steady-state accumulation of SNAP-25
on internal membranes, as we and others (9-13) have routinely observed
that at steady state, wild-type SNAP-25 is membrane-associated, whereas
SNAP-25 palmitoylation mutants are cytosolic. To reconcile these
steady-state data with our kinetic data examining the initial
association of SNAP-25 with membranes, we propose the following model
(Fig. 8). Following the
post-translational assembly of the SNAP-25·syntaxin t-SNARE complex
in the cytosol, the complex is targeted to membranes and is tethered
there by the transmembrane domain of syntaxin (Fig. 8, step
1). Our data showing that newly synthesized wild-type SNAP-25 and
the SNAP-25 palmitoylation mutant traffic to membranes equally well in
the presence of syntaxin are in excellent agreement with this model.
Once on the membrane, syntaxin-associated SNAP-25 is accessible to
palmitoylacyltransferase and is palmitoylated (Fig. 8, step
2), a process that begins ~20 min after the synthesis of the
SNAP-25 polypeptide (12).
In much the same way that the initial membrane association of SNAP-25
is mediated by its binding to syntaxin, the initial association of the
non-myristoylated G protein Steady-state metabolic radiolabeling studies have revealed that most
SNAP-25 in PC12 cells is not bound to any syntaxin.2 Since
most SNAP-25 is palmitoylated and membrane-associated in these cells at
steady state (Refs. 11 and 12 and this study), it is likely that
SNAP-25·syntaxin complexes dissociate on membranes. This process may
either occur spontaneously or be catalyzed by the action of an
unfolding enzyme such as the N-ethylmaleimide-sensitive fusion protein. Therefore, even if palmitoylated SNAP-25
dissociates from syntaxin, it will remain stably associated with
membranes (Fig. 8, step 3). If, on the other hand, SNAP-25
in the complex was not previously palmitoylated (as in cells expressing
the palmitoylation mutant of SNAP-25), dissociation from syntaxin may
result in the liberation of SNAP-25 into the cytosol (Fig. 8,
step 4). The data presented in this study and in the
numerous cited studies are in excellent agreement with this kinetic
model of SNAP-25 trafficking to membranes.
Although endogenous SNAP-25 is usually palmitoylated and
membrane-associated, there are other members of the SNAP-25 family that
are membrane-associated despite the lack of demonstrated palmitoylation
or a putative palmitoylation domain. The SNAP-25 homolog in yeast,
Sec9p, is not fatty-acylated, yet is still found tightly associated
with membranes (31). Similarly, the recently described mammalian
SNAP-25 homolog SNAP-29 does not possess a palmitoylation domain, yet
>50% of the molecule is associated with membranes (32, 33). In
characterizing SNAP-29, a possible role of syntaxin in the membrane
association of SNAP-29 was proposed. Finally, even after chemical
deacylation, SNAP-25 remains tightly associated with membranes, and
deacylated SNAP-25 remains bound to syntaxin (12), suggesting that
syntaxin binding alone is sufficient to tether SNAP-25 to membranes.
In conclusion, we have shown that the initial association of SNAP-25
with intracellular membranes is mediated by its association with
syntaxin. It is the hydrophobic transmembrane domain of syntaxin that
brings SNAP-25 to membranes, and we propose that it is only after
syntaxin-mediated tethering to membranes that SNAP-25 is palmitoylated
by palmitoyltransferase. The data presented in this study provide a
model with which to begin to address the precise mechanism by which
t-SNARE assembly proceeds in vivo.
We thank Drs. Juan Bonifacino, Robert
Wenthold, Richard Scheller, Reinhard Jahn, and Michael Veit for the
generous gifts of reagents and David Winkler for oligonucleotide
synthesis and automated DNA sequence analysis. We also thank Dr. Dinah
Singer and all members of the Roche laboratory for valuable discussions
and critical reading of the manuscript.
*
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.
§
Present address: Inst. Pasteur, U277, Biologie Moleculaire du Gen,
25 rue du Dr. Roux, 75724 Paris Cedex 15, France.
¶
To whom correspondence should be addressed: NCI, NIH, Bldg.
10, Rm. 4B36, Bethesda, MD 20892. Tel.: 301-594-2595; Fax:
301-496-0887; E-mail: paul.roche@nih.gov.
2
P. A. Roche, unpublished observations.
The abbreviations used are:
t-SNARE, target
SNARE;
PAGE, polyacrylamide gel electrophoresis.
Targeting of SNAP-25 to Membranes Is Mediated by Its
Association with the Target SNARE Syntaxin*
,§, and
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
i1 subunit require either myristoylation or, in the case
of a myristoylation-defective mutant of Gi1, binding to
membrane-associated G protein 
and 
subunits (16). In each of
these examples, the initial association with membranes is essential for
subsequent palmitoylation of the substrate, as cellular
palmitoyltransferases are not cytosolic and have been exclusively
localized to intracellular membranes (17-19).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) (10) was obtained from
Dr. Michael Veit (Freie Universitat Berlin, Berlin, Germany) and was
subcloned into the EcoRI site of pcDNA3. The
transmembrane mutant of syntaxin 1A (encoding amino acids 1-266,
termed syntaxin ) was generated using the polymerase chain reaction
by the introduction of a stop codon in the reverse primer. The sequence
of the construct was verified by DNA sequence analysis. The cDNA
encoding Tac in the vector pCDM8 and the anti-Tac monoclonal antibody
7G7 were obtained from Dr. Juan Bonifacino (NICHD, National Institutes
of Health). The anti-SNAP-25 monoclonal antibody was obtained from
Sternberger Monoclonals (Baltimore, MD), and the anti-syntaxin 1A
monoclonal antibody was from Wako Bioproducts (Richmond, VA). The
anti-calnexin antibody was the generous gift of Dr. Robert Wenthold
(NIDCD, National Institutes of Health). The anti-VAMP monoclonal
antibody C1 10.1 was obtained from Dr. Reinhard Jahn (Max Planck
Institute for Biophysical Chemistry, Göttingen, Germany).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
SNAP-25 and syntaxin associate in the cytosol
of PC12 cells. A, PC12 cells were pulse-labeled with
[35S]methionine for 15 min and either harvested
immediately (pulse) or incubated for 3 h in complete
medium after the pulse labeling (chase). The cells were
lysed, and control, syntaxin, or SNAP-25 immunoprecipitations were
performed. The amount of SNAP-25 present in each immunoprecipitate was
analyzed by SDS-PAGE and fluorography. B, PC12 cells were
labeled with [35S]methionine for 1 h and homogenized
using a ball-bearing homogenizer, and cytosolic (C) and
membrane (M) fractions were obtained following
centrifugation at 110,000 × g as described under
"Experimental Procedures." Following immunoprecipitation using
control, anti-SNAP-25, and anti-syntaxin antibodies, the relative
amounts of SNAP-25 in equivalent portions of the cytosolic and membrane
fractions were analyzed by SDS-PAGE and fluorography. C, the
isolated cytosolic and membrane fractions of PC12 cells were
immunoblotted with antibodies specific for endogenous calnexin,
syntaxin 1, SNAP-25, or VAMP.
View larger version (26K):
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Fig. 2.
SNAP-25 and syntaxin binding precedes
membrane association. HeLa cells expressing SNAP-25 alone
(A) or SNAP-25 and wild-type syntaxin
(B) were pulse-labeled with
[35S]methionine for 15 min and chased for 3 h. At
each time point, the cells were harvested, homogenized using a
ball-bearing homogenizer, and separated into cytosolic (C)
and membrane (M) fractions. Following immunoprecipitation
with SNAP-25-specific antibodies, the relative amounts of each protein
in equivalent portions of the cytosolic and membrane fractions were
analyzed by SDS-PAGE and fluorography.
-chain, were subjected to the same
pulse-chase and subcellular fractionation analyses described above.
Unlike the t-SNARE proteins described above, 35S-labeled
Tac was found exclusively in the membrane fraction of transfected HeLa
cells both after the pulse labeling as well as after a 3-h chase (data
not shown). Since Tac is an integral membrane protein like syntaxin and
was not detected in the cytosolic fraction, these data demonstrate that
the isolation of cytosolic SNARE complexes was not an artifact of transfection.
View larger version (12K):
[in a new window]
Fig. 3.
Association of SNAP-25 with membranes is
enhanced by syntaxin. Densitometry was used to quantitate the
amount of newly synthesized SNAP-25 present in the cytosolic
(C) and membrane (M) fractions of HeLa cells
transfected with either SNAP-25 alone or with SNAP-25 and wild-type
syntaxin 1. The amount of 35S-labeled SNAP-25 present in
each sample was expressed as a percentage of the total amount of
35S-labeled SNAP-25 recovered under each condition.
Open bars indicate the subcellular distribution of SNAP-25
following a 15-min pulse labeling, and solid bars indicate
the subcellular distribution of SNAP-25 following a 3-h chase. The data
represent the average of two independent experiments.
) resided exclusively in the cytosol (Fig.
4). Identical results were obtained in
anti-syntaxin immunoblot analysis of the isolated fractions (data not
shown).
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Fig. 4.
The transmembrane domain of syntaxin is
required for membrane association of the t-SNARE complex. HeLa
cells transfected with cDNAs encoding either wild-type syntaxin
(upper panels) or syntaxin (lower panels)
were pulse-labeled with [35S]methionine for 1 h,
chased for 3 h in complete medium, and subjected to subcellular
fractionation to generate cytosolic (C) and membrane
(M) fractions. Immunoprecipitation using an anti-syntaxin
monoclonal antibody revealed that wild-type syntaxin resides
exclusively in the membrane fraction, whereas syntaxin resides
exclusively in the cytosol.
with a mutant of SNAP-25 that lacked all potential
palmitoylation sites (SNAP-25) resulted in efficient assembly of the
t-SNARE complex (Fig. 5B); however, in this case, the
SNAP-25·syntaxin complexes were exclusively cytosolic and were not
present in the membranes even after a 3-h chase. These data demonstrate
that efficient assembly of t-SNARE complexes in vivo does
not require the presence of the syntaxin transmembrane domain.
Furthermore, these data show that the transmembrane domain of syntaxin
and/or the palmitoylation domain of SNAP-25 is required for the
association of t-SNARE complexes with membranes.
View larger version (31K):
[in a new window]
Fig. 5.
SNAP-25 association with membranes is
regulated by its binding to syntaxin. HeLa cells transfected with
cDNA encoding wild-type SNAP-25 and wild-type syntaxin
(A), SNAP-25 and syntaxin (B), or wild-type SNAP-25 and syntaxin (C) were pulse-labeled with
[35S]methionine for 15 min and chased for 3 h.
Following the 3-h chase, the cells were harvested, homogenized using a
ball-bearing homogenizer, and separated into cytosolic (C)
and membrane (M) fractions. Following immunoprecipitation
with antibodies specific for SNAP-25 and syntaxin, the relative amounts
of SNAP-25 and syntaxin in the cytosolic and membrane fractions were
analyzed by SDS-PAGE and fluorography.
-containing
t-SNARE complexes to membranes. Surprisingly, although t-SNARE complex
assembly proceeded normally, the t-SNARE complexes remained exclusively
in the cytosol (Fig. 5C). This study also demonstrated that
the ability of syntaxin to enhance SNAP-25 association with membranes
was not merely a consequence of its binding to SNAP-25. The extent of
SNAP-25 association with membranes under each condition described above
was quantitated by PhosphorImager analysis and is shown in Fig.
6. Since wild-type SNAP-25 efficiently associated with membranes in cells expressing wild-type syntaxin, but
did not associate with membranes in cells expressing syntaxin , we
conclude that the palmitoylation domain of SNAP-25 alone is not
sufficient to serve as the primary targeting signal for the initial
association of SNAP-25 with membranes.
View larger version (9K):
[in a new window]
Fig. 6.
Membrane association of SNAP-25 is regulated
by syntaxin. Densitometry was used to quantitate the amount of
newly synthesized SNAP-25 present in the cytosolic (C) and
membrane (M) fractions of HeLa cells transfected with
cDNA encoding wild-type SNAP-25 and wild-type syntaxin (left
panel), SNAP-25 and syntaxin (center panel), or
SNAP-25 and syntaxin (right panel) following a 3-h
chase. The amount of 35S-labeled SNAP-25 present in each
sample is expressed as a percentage of the total amount of
35S-labeled SNAP-25 recovered under each condition.
, we reasoned that the intact transmembrane domain of syntaxin may be required for the efficient association of newly synthesized SNAP-25
with membranes. To test this directly, we coexpressed wild-type
syntaxin with the SNAP-25 palmitoylation mutant SNAP-25. In
agreement with our hypothesis, we found that t-SNARE complexes containing SNAP-25 associated with membranes equally well as wild-type SNAP-25 after a 3-h chase (Fig.
7). These data demonstrate that the
presence of the palmitoylation domain of SNAP-25 is not necessary for
SNAP-25·syntaxin t-SNARE complex association with membranes and show
that the initial membrane anchoring of the complex is dependent on the
transmembrane domain of syntaxin.
View larger version (17K):
[in a new window]
Fig. 7.
The transmembrane domain of syntaxin is
responsible for the association of SNAP-25 with membranes. HeLa
cells coexpressing wild-type syntaxin and SNAP-25 were labeled with
[35S]methionine for 15 min and chased for 3 h.
Following the 3-h chase, the cells were harvested, homogenized using a
ball-bearing homogenizer, and separated into cytosolic (C)
and membrane (M) fractions. Following immunoprecipitation
with antibodies specific for SNAP-25 and syntaxin, the relative amounts
of SNAP-25 and wild-type syntaxin in the cytosolic and membrane
fractions were analyzed by SDS-PAGE and fluorography.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (14K):
[in a new window]
Fig. 8.
Model of SNAP-25 association with
membranes. Following the post-translational assembly of the
SNAP-25·syntaxin t-SNARE complex in the cytosol, the complex is
targeted to membranes by virtue of the presence of the transmembrane
domain of syntaxin (step 1). Once on membranes, SNAP-25
(S-25) is palmitoylated by membrane-associated
palmitoyltransferases (PAT; step 2), thereby
generating the steady-state SNAP-25·syntaxin t-SNARE complex. Even if
palmitoylated SNAP-25 dissociates from syntaxin (Stx) at
this point (step 3), SNAP-25 will remain stably associated
with membranes. However, if non-palmitoylated SNAP-25 dissociates from
syntaxin (step 4), SNAP-25 will diffuse into the
cytosol.
i1 with membranes is
mediated by its binding to membrane-anchored G protein and subunits (16). Like SNAP-25, Gi1 is palmitoylated only
after membrane tethering (16). In addition, palmitoylation of GAD65 requires prior targeting to intracellular membranes, leading Solimena et al. (30) to propose that for GAD65 and other
palmitoylation substrates, palmitoylation alone may not be the primary
membrane targeting signal. Furthermore, palmitoylation of SNAP-25
requires an intact secretory pathway (12), suggesting that like other palmitoyltransferase substrates, SNAP-25 is palmitoylated following arrival at the plasma membrane. Since all palmitoylacyltransferase activity has been localized to intracellular membranes and most of this
activity is thought to reside at the plasma membrane (14, 17-19), our
model offers a mechanism by which newly synthesized, non-palmitoylated
SNAP-25 would be brought into the proximity of this membrane-bound enzyme.
ACKNOWLEDGEMENTS
FOOTNOTES
These authors contributed equally to this work.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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