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INTRODUCTION |
SNAP-25 homologues belong to a small family of structurally
similar proteins thought to function in neurotransmission and axonal
growth (1-5). SNAP-25 is very abundant in neurons where it localizes
to presynaptic membranes and synaptic vesicles (1, 6). It has been
shown that SNAP-25 and syntaxin-1, two proteins localized at the plasma
membrane, form a complex with VAMP-2, an integral membrane protein of
synaptic vesicles (3). This heterotrimeric complex is a receptor
(SNARE) for SNAP and NSF, two soluble factors required for vesicular
traffic (3). A current model predicts that SNARE receptors at an
acceptor compartment (t-SNAREs) interact in a "lock and key"
fashion with SNARE receptors at the surface of transport vesicles
(v-SNAREs) (reviewed in Ref. 7). Recently, it has been proposed that
SNARE components, by themselves, are sufficient to mediate fusion
between two lipid bilayers (8) and pairing of specific v-SNAREs and
t-SNAREs determines specificity of organelle traffic. In agreement with this hypothesis, there are multiple members of the VAMP and syntaxin family of proteins, each localized at a specific intracellular compartment (9). A homolog of SNAP-25, called SNAP-23, was identified
from a human B lymphocyte library by yeast double-hybrid screening
(10). SNAP-23 binds tightly to several syntaxins and VAMPs in
vitro (10). Whether individual syntaxin, VAMP-2, and SNAP-25
homologs work in specific exocytotic events is still unknown.
We cloned syndet, which is 87% identical to human SNAP-23 from a mouse
adipocyte library (11). Syndet and SNAP-23 are expressed in many
tissues (11-14). Syndet and syntaxin-4 are localized at the plasma
membrane of adipocytes (11, 12, 14) and VAMP-2 is localized in glucose
transporter-containing vesicles (14-16). It has been proposed that the
SNARE complex, syndet·syntaxin-4·VAMP-2 mediates
insulin-dependent exocytosis of glucose
transporter-containing vesicles to the plasma membrane of adipocytes
(17). Thus, syndet in adipocytes, like SNAP-25 in neurons, seems to be
involved in exocytosis of intracellular vesicles at the cell surface.
SNAP-25 and syndet are integral membrane proteins, but do not have any predicted signal sequence or transmembrane domains (1, 11). Post-translational modifications may provide these proteins with a
hydrophobic anchor to membranes. It has been shown that SNAP-25 is
palmitoylated through a thioester bond to some or all of its four
cysteines clustered at a region near the center of the protein sequence
(18, 19). Deletion of this region abolishes palmitoylation of SNAP-25
and its binding to membranes (20). Also point mutations at single
cysteines of SNAP-25 largely inhibit palmitoylation and membrane
binding (19). This study was designed to investigate which domain of
syndet is critical for its intracellular localization. In this paper we
show that the number and configuration of cysteines at the
cysteine-rich domain act as major determinants for the subcellular
distribution of syndet.
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EXPERIMENTAL PROCEDURES |
Syndet Mutagenesis--
The original template for polymerase
chain reactions (PCR)1 of
syndet was pcDNA1-syndet plasmid (11). Mutations were
encoded in the oligonucleotide primers synthesized by Life
Technologies, Inc. (Gaithersburg, MD). The enzyme used for PCR was
Pwo DNA Polymerase (3 units/reaction, Boehringer Mannheim
Corp.) with its supplied reaction buffer. The primers were used at a
final concentration of 2 µM. The deoxynucleotide
triphosphates (Promega, Madison, WI) were used at a final concentration
of 200 µM. The PCR was carried out on a "GeneAmp PCR
System 2400" (Perkin-Elmer, Applied Biosystems Division, Foster City,
CA). All restriction enzymes were purchased from Promega.
Syndet cDNA was amplified from pcDNA1-syndet using
primers: TGCAATCAGTACAAGCTTGAGAACTGTGGAGGCTGGAGAGGAGTG (number
1) which encodes a HindIII site, and
TCAGGTCACTCTAGACTACTTATACAGCTGCTTCTT (number 2) which encodes an
XbaI site. It was then subcloned into the
HindIII-XbaI sites of vector pCB7 (21) to obtain
syndet-pCB7.
Delta-syndet mutant was also made from pcDNA1-syndet in
two separate PCR reactions, using primers 1 and
CGCATCTTACCCGGGCTTGTTGAGTTCTGTTAAAGTCTT, which encodes an
XmaI site, for the first reaction, and primers 2 and
AGTGTTACGGTACCCGGGAATAGGACAAAGAACTTTGAG, which also encodes an
XmaI site, for the second. The products of the first PCR
reaction were digested with the restriction enzymes HindIII
and XmaI and the products of the second PCR reaction with
XmaI and XbaI. These two products were ligated
into pCB7 vector to obtain delta-syndet-pCB7.
Syndet C2S/C4S/C5S--
pCB7 vector was made using the technique
of "overlap extension" PCR (22) with syndet-pCB7 as the
template. Primers 1 and GGAAATACACAAACCAGAACACTTGTTGAG were used to
make the 5'-half of syndet C2S/C4S/C5S and primers 2 and
TTGTGTATTTCCCCGTCTAATAGGACAAAG to make the 3'-half. The two products
were combined in a second PCR, digested with HindIII and
XbaI, and subcloned into pCB7.
All other mutants were created using a combination of "long distance
inverse" PCR (23) and "touchdown" PCR methods (24) with
syndet-pCB7 as the template. The two primers were designed to encode the mutations and align "back to back" on the
syndet-pCB7 backbone. The reverse primer was phosphorylated
using T4 polynucleotide kinase (Promega) in the supplied reaction
buffer along with the addition of ATP (2 mM). The PCR
buffer was the same as above, except that it contained
N,N-dimethylformamide at a final concentration of 1% (v/v).
The PCR parameters were the following: denaturation at 94 °C for
30 s, annealing at 55 °C for 30 s, and extension at
72 °C for 6 min. The annealing temperature was then reduced by
1 °C per cycle for the next 11 cycles, and then kept at 44 °C for
the following 30 cycles. The PCR products were treated with
DpnI, to digest the template DNA, purified by low-melt
agarose gel electrophoresis, and ligated with T4 DNA-ligase. Primers
TGCATCAGTCCTTGTAATAGGACA and GAGGCCTGAACACTTGTTGAGTTC were used
for making syndet C2S/C4S-pCB7; ATCTGCCCTTCCAATAGGACAAAGAAC and
GCAGAGGCCACTACACTTGTTGAGTTC for making syndet
C2S/C5S-pCB7; GCAGAGGCCACTACACTTGTTGAGTTC and
CCTTCAAATAGGACAAAGAAC for making syndet C4S/C5S-pCB7;
CTCTCCATCTGCCCTTGTAAT and CCCACAAGACTTGTTGAGTTCTGT for syndet
C1S/C3S-pCB7; CTCTGCATCTCCCCTTGTAATAGGACA and
CCCACAACACTTGTT for syndet C4S-pCB7;
CCCACAACACTTGTT and CTCTGCATCTGCCCTTCTAATAGGACAAAGAAC for syndet
C5S-pCB7; TCCTCACTTCTGTATTTGCTG and TCCGCAAAGAAACTCATT for
syndet delta-BoNT/E; CTCTGCATCTGCCCTTGTAATAAGGCAACATGGGGA and CCCACAACACTTGTTCAGCATAGTGATGGT for syndet

1. The identity of all the mutants was confirmed
by restriction analyses and sequencing.
Cell Culture and Transfection--
AtT-20 cells were cultured
and transfected as described (25).
Immunofluorescence and Confocal Microscopy--
AtT-20 cells
were grown on poly-D-lysine-coated glass coverslips and
treated for immunofluorescence as described (11). As primary
antibodies, anti-syndet affinity purified rabbit polyclonal antibodies
at 1:80 dilution (11); mouse monoclonal anti-SNAP 25 SMI-81 antibodies
from Sternberger Monoclonals Inc. (Baltimore, MD) at 1:400 dilution;
and anti-ACTH monoclonal antibody, clone 57 from Research Diagnostics
Inc. (Flanders, NJ), at 1:100 were used. Donkey anti-rabbit
Cy3-conjugated antibodies and goat anti-mouse fluorescein
isothiocyanate-conjugated antibodies (Jackson Immunoresearch Chemicals,
West Grove, PA) were used at 1:200 dilution. The immunofluorescence images were obtained at the Confocal Microscopy Facility of the Herbert
Irving Comprehensive Cancer Center at the Columbia Presbyterian Medical
Center. A Zeiss Axiovert 100TV fluorescence microscope (Carl Zeiss,
Thornwood, NY) with a Zeiss LSM410 laser scanning confocal attachment
was used to obtain the images. The cells were excited with an
Argon-Krypton laser using the standard wavelengths for rhodamine and
fluorescein isothiocyanate. The images were collected as 1-µm thick
optical sections. They were processed using Zeiss LSM 3.95 software and
Adobe Photoshop 3.0 (Adobe Systems).
Immunoelectron Microscopy--
Immunogold labeling of broken and
agarose-embedded AtT-20 cells was done as described elsewhere (25).
Rabbit polyclonal anti-syndet antibodies were used at 1:20 dilution and
mouse monoclonal anti-SNAP-25 antibodies were used at 1:100 dilution.
Colloidal gold donkey anti-rabbit IgG (12 nm) and colloidal gold donkey anti-mouse IgG (6 nm) were used for immunolabeling at a concentration of 1:100. Immunolabeled cells were osmicated (0.5% for 30 min), dehydrated, and embedded in tEPON (Tousimis Research Corp., Rockville MD). Thin sections (70 nm) were cut on an AO Ultracut microtome (Reichert-Jung, Vienna, Austria), counterstained with uranyl acetate and lead citrate, and viewed and photographed on a JEOL 1200EX electron
microscope (JEOL USA, Inc., Peabody MA).
Cell Fractionation and Western Blots--
Protease inhibitors,
leupeptin (6 µg/ml) and aprotinin (1.5 µg/ml), were purchased from
Boehringer Mannheim, Phenylmethylsulfonyl fluoride (2 mM)
was from Sigma. Cells were grown on 6-cm plates for at least 72 h
and homogenized in 400 µl of buffer A (10 mM Tris-Cl, pH
7.4, 150 mM NaCl, 2 mM EDTA, and protease
inhibitors) by passing the cells through an insulin needle six times.
The homogenates were mixed with an equal volume of SDS sample buffer, boiled, and loaded into the SDS-PAGE gel. In some experiments, a
fraction of the homogenate (200 µl) was spun at 400,000 × g for 30 min. The supernatant fraction was recovered and the
pellet was resuspended in 200 µl of buffer A. Each fraction (40 µl
each) was mixed with an equal volume of SDS sample buffer, boiled, and loaded into the SDS-PAGE gel. Further steps including electrophoresis and Western blotting were carried out as described previously (11).
Metabolic Labeling and Immunoprecipitation of Syndet--
Cells
expressing syndet, delta-syndet, and syndet C1S/C3S were
labeled with ([3H]palmitate (0.5 mCi/ml, NEN Life Science
Products Inc., Boston, MA) for 2 h, as described. (19). Cells
grown in a 3.5-cm plate were washed twice with buffer A and lysed in
1.0 ml of buffer A with Nonidet P-40 (1% v/v, Sigma) along with
protease inhibitors. The homogenate was centrifuged for 10 min at
5,000 × g. Affinity purified syndet antibody (10 µl)
was added to the supernatants and samples were incubated at 4 °C
with gentle agitation for 1 h. Protein A-Sepharose beads (40 µl
dry beads, Boehringer Mannheim) were added to the samples, which were
further incubated at 4 oC for 45 min while being agitated.
After centrifugation, pelleted immunobeads were washed three times with
lysis buffer A and mixed with SDS-PAGE sample buffer. Samples were
boiled and analyzed by SDS-PAGE electrophoresis and fluorography as
described (20). Preliminary experiments were run to determine that
syndet, delta-syndet, and syndet C1S/C3S were
immunoprecipitated to the same extent by affinity purified syndet
antibodies. To visualize immunoprecipitated wild-type and mutated
syndet by Western blot, immunobeads were mixed with sample buffer
without reducing agents.
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RESULTS |
Targeting of Exogenous Syndet in Transfected AtT-20 Cells--
The
endogenous level of syndet protein in AtT-20 cells is undetectable by
Western blot and immunofluorescence (see below). We determined the
distribution of exogenous syndet in stably transfected AtT-20 cells by
immunofluorescence (Fig. 1). Syndet
immunoreactivity was seen along the plasma membrane of the cell body,
the neurite-like processes and the tips (Fig. 1B). Staining
for SNAP-25, like syndet, was predominant along the entire plasma
membrane (Fig. 1A). However, SNAP-25 antibodies also stained
a region near the nucleus and the cytoplasm within the tip. We have
shown elsewhere by immunoelectron microscopy that endogenous syndet in
adult mouse kidney is found predominantly at the plasma membrane (11).
At the ultrastructural level, we confirmed that exogenous syndet in
AtT-20 cells was indeed localized at the plasma membrane. Fig.
2 shows that exogenous syndet (12-nm gold
particles) and endogenous SNAP-25 (6-nm gold particles) are localized
at the plasmalemma in agarose-embedded broken cells. There was no
syndet or SNAP-25 labeling associated with the nuclear membrane, or
mitochondria or endogenous murine leukemia virus particles. These
results indicate that exogenous syndet expressed in AtT-20 cells is
able to localize at the plasmalemma. We conclude that AtT-20 cells can
be used as a model to study the role of various syndet domains in
plasma membrane localization.

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Fig. 1.
Double immunofluorescence localization of
endogenous SNAP-25 and exogenous syndet. Confocal
immunofluorescence microscopy of stably transfected AtT-20 cells
expressing wild-type syndet. Cells were double-stained with mouse
monoclonal antibodies against SNAP-25 and fluorescein
isothiocyanate-conjugated antibodies against mouse IgG (A)
and with rabbit polyclonal antibodies against syndet and Cy3-conjugated
antibodies against rabbit IgG (B). The cell body is
indicated by arrowheads, the tips by
arrows.
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Fig. 2.
Immunogold staining of broken cells
expressing wild-type syndet. Syndet (12 nm gold) and SNAP-25 (6 nm
gold) are detected at the plasma membrane. There is no staining of
mitochondria (m), endogenous murine leukemia virus
(v), or nuclear membrane (not shown). Bar, 100 nm.
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Domains of Syndet Involved in Plasma Membrane Localization--
We
prepared three deletion mutants of syndet to find domains responsible
for targeting syndet to the plasma membrane (Fig. 3, upper panel). The deletion
mutant delta-syndet lacks the syndet amino acid sequence from
Cys79 to Cys85. Pro86 was left
unchanged, and Cys87 was mutated to a glycine. Thus,
delta-syndet does not have any cysteines. Syndet 
1 lacks the
amino-terminal region which has a high propensity to form coiled-coil
structures (11) and a short sequence after the cysteine-rich domain.
The cysteine-rich domain was entirely preserved in the syndet 
1
mutant. Syndet delta-BoNT/E is the expected proteolysis product of
mouse syndet protein treated with the botulinum toxin E (BoNT/E) (26).
Syndet delta-BoNT/E lacks approximately 40% of the carboxyl-terminal region with high probability to form coiled-coil structures (11). Western blots of homogenates from AtT-20 cells expressing wild type
syndet (26 kDa), delta-syndet (25 kDa), syndet 
1 (20 kDa), and
syndet delta-BoNT/E (23 kDa) are shown in Fig. 3, A-C.

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Fig. 3.
Deletion mutants of syndet. The
upper part of Fig. 3 shows the deletion mutants of syndet
used in this work. The cysteine-rich domain and the regions predicted
to form coiled-coil conformation are indicated. The lower
part of the figure illustrates Western blots of wild-type and
mutant syndet proteins. Homogenates of AtT-20 cells expressing
wild-type syndet (A-C), delta-syndet (A), syndet
 1 (B), and syndet-delta BoNT/E (C), were
mixed with sample buffer and loaded onto SDS-PAGE. Blots were probed
with affinity purified syndet antibody (1:400). Bands were visualized
by enhanced chemiluminescence.
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Deletion of the entire cysteine-rich domain of syndet (delta-syndet)
shifted the distribution of syndet immunoreactivity from the plasma
membrane to a diffuse intracellular localization (Fig. 4B). The localization of
endogenous SNAP-25 was not changed by expression of delta-syndet (Fig.
4A). We conclude that the cysteine-rich domain of syndet is
necessary for targeting the protein to the cell surface. Syndet 
1
and syndet delta-BoNT/E immunofluorescence staining accumulated at the
plasma membrane of the cell body (Fig. 4, C and
E), of the neurite-like processes (not clearly visible in
the optical sections shown in Fig. 4) and of the tips (Fig. 4, C and D). Thus, the subcellular distribution
of syndet 
1 and syndet delta-BoNT/E was similar to the
distribution of exogenous wild-type syndet (compare with Fig. 1). These
experiments indicate that the entire predicted
-helix at the amino
terminus of syndet and at least 40% of the helix at the carboxyl
terminus are not required for targeting syndet to the plasma membrane.
Unlike these regions, the cysteine-rich domain is necessary for plasma
membrane targeting.

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Fig. 4.
Confocal immunofluorescence microscopy of
stably transfected AtT-20 cells expressing different deletion mutants
of syndet. Cells expressing delta-syndet (A and
B) were double-stained with antibodies against SNAP-25
(A) and against syndet (B) as indicated in the
legend to Fig. 1. Cells expressing syndet  1 (C) and
syndet-delta BoNT/E (D and E) were stained with
antibodies against syndet.
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Requirement of Cysteines for Syndet Localization at the Plasma
Membrane--
The cysteine-rich domains of wild-type syndet and two
alternatively spliced isoforms of SNAP-25, SNAP-25 A and SNAP-25 B (27) are shown in Fig. 5. The five cysteines
of syndet are designated as Cys1, Cys2,
Cys3, Cys4, and Cys5 and correspond
to Cys79, Cys80, Cys83,
Cys85, and Cys87, respectively. Unlike syndet,
SNAP-25 A and SNAP-25 B have four cysteines in their cysteine-rich
domain. In SNAP-25 A, the cysteine corresponding to Cys3 of
syndet is substituted by a phenylalanine. In SNAP-25 B, the cysteine
corresponding to syndet Cys1 is substituted by a
phenylalanine. The triplet of cysteines, -Cys2-Cys4-Cys5-, of syndet is
conserved in both SNAP-25 A and SNAP-25 B. To study the role of
individual cysteines at the cysteine-rich domain, we prepared a series
of mutations. In the mutant syndet C2S/C4S/C5S, all the cysteines of
the -Cys2-Cys4-Cys5- triplet were
substituted with serines. Within the
-Cys2-Cys4-Cys5- triplet, we also
made pairwise substitutions, syndet C2S/C4S, syndet C2S/C5S, syndet
C4S/C5S, of two cysteines with serines. In syndet C4S/C4S and syndet
C5S, Cys4 and Cys5, respectively, were
substituted with serines. In syndet C1S/C3S, both Cys1 and
Cys3 of syndet were substituted with serines.
Immunofluorescence experiments were performed on AtT-20 cells
transiently expressing either wild type or mutated syndet proteins. Plasma membrane staining of wild-type syndet in transiently transfected AtT-20 cells was indistinguishable from that observed in the stable AtT-20 cell lines. Syndet C2S/C4S/C5S was found in the cytoplasm (Fig.
6A), and had a distribution
similar to delta-syndet (Fig. 4B). This experiment indicates
that the conserved -Cys2-Cys4-Cys5-
triplet is critical for syndet localization at the plasma membrane. Syndet C4S/C5S (Fig. 6B), syndet C2S/C4S (Fig.
6C), and syndet C2S/C5S (Fig. 6D) were also found
in the cytoplasm and did not localize at the plasma membrane. Syndet
proteins with single point mutations at C5S (Fig. 6E) or C4S
(Fig. 6F) were localized at the plasma membrane. Thus, a
mutation of any pair of cysteines within the conserved triplet is
sufficient to prevent plasma membrane localization. Single
substitutions do not alter syndet distribution. We conclude that at
least 2 cysteines within the
-Cys2-Cys4-Cys5- triplet are
necessary for localization of syndet at the plasma membrane.

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Fig. 6.
Confocal microscopy of transiently
transfected AtT-20 cells expressing wild-type and mutant syndet
proteins. Cells expressing syndet C2S/C4S/C5S (A),
syndet C4S/C5S (B), syndet C2S/C4S (C), syndet
C2S/C5S (D), syndet C5S (E), and syndet C4S
(F) were stained with antibodies against syndet as indicated
in Fig. 1.
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Subcellular Localization of Syndet C1S/C3S--
Syndet
distribution in AtT-20 cell lines stably expressing the syndet C1S/C3S
protein was examined. In contrast to wild-type syndet (Fig.
1B), the mutant, syndet C1S/C3S, was found at an intracellular site as well as at the plasma membrane (Fig.
7B). Double staining with
SNAP-25 antibodies indicate that endogenous SNAP-25 is predominantly at
the plasma membrane (Fig. 7A). We determined the relative
distribution of ACTH and syndet immunoreactivities in AtT-20 cells
expressing wild-type syndet and syndet C1S/C3S (not shown). ACTH
immunoreactivity is concentrated in a Golgi-like region near the
nucleus and at the tips (25) where ACTH-containing granules accumulate
(28). Wild-type syndet did not co-localize with ACTH immunoreactivity
either at the cell body or at the tips. Syndet C1S/C3S co-localized
with ACTH immunoreactivity in the Golgi-like compartment, but not at
the tips of the processes. These results show that mutations at
Cys1 and Cys3: (a) shift a fraction
of syndet from its plasma membrane localization to a Golgi-like
intracellular compartment; (b) do not induce re-distribution of syndet to dense core granules.

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Fig. 7.
Confocal microscopy of stably transfected
AtT-20 cells expressing syndet C1S/C3S. Double immunofluorescence
staining with antibodies against SNAP-25 and syndet was as indicated in
Fig. 1.
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Role of the Cysteine-rich Domain in Syndet Binding to
Membranes--
AtT-20 cells expressing wild-type syndet, delta-syndet,
and syndet C1S/C3S were homogenized in a buffer containing either 150 mM NaCl or 100 mM
Na2CO3 at pH 11.5. Buffers containing sodium carbonate at pH 11.5 are known to dissociate peripheral proteins from
membranes (29). The homogenates were then centrifuged at 400,000 × g for 30 min to obtain a membrane-containing pellet and a
cytosol-containing supernatant. Wild type SNAP-25 and syndet were
mostly or entirely recovered in the pellet fraction (Fig. 8). We conclude that exogenous syndet in
AtT-20 cells is an integral membrane component, like endogenous syndet
in adipocytes. Overexpression of syndet did not affect the binding of
endogenous SNAP-25 to membranes (Fig. 8). Delta-syndet was almost
entirely recovered in the supernatant fraction, indicating that
deletion of the cysteine-rich domain makes the protein soluble (Fig.
8). Approximately 50% of syndet C1S/C3S was tightly associated to
membranes (Fig. 8). We conclude that Cys1 and
Cys3 are important, but not absolutely necessary, for
syndet binding to membranes.

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Fig. 8.
Western blot of fractions from AtT-20 cells
expressing wild-type and mutant syndet proteins. AtT-20 cells
expressing wild-type syndet, delta-syndet, and syndet C1S/C3S were
grown in 6-cm plates. Cells were homogenized in 400 µl of the
following buffers: 10 mM Tris-HCl, 1 mM EDTA,
150 mM NaCl, pH 7.4 (upper panel); 0.1 M sodium carbonate, pH 11.5, 1 mM EDTA
(lower panel). Buffers contained protease inhibitors as
indicated under "Experimental Procedures." Equal volumes of
homogenates (H), membrane-containing pellets (P),
and cytosol-containing supernatants (S) were prepared as
described under "Experimental Procedures." Blots were probed with
antibodies against syndet and SNAP-25. This experiment was repeated
three times with similar results.
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Syndet Is Palmitoylated--
To determine whether syndet is
palmitoylated, we incubated transfected AtT-20 cells expressing
wild-type syndet, delta-syndet, and syndet C1S/C3S with
[3H]palmitic acid. Palmitoylated proteins were analyzed
by immunoprecipitation and SDS-PAGE fluorography (Fig.
9). Wild-type syndet and syndet C1S/C3S,
but not delta-syndet, incorporated labeled palmitate. These experiments
and the ones in Fig. 8 indicate that the cysteine-rich motif of syndet
is palmitoylated and that incorporation of fatty acid is necessary for
syndet binding to membranes.

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Fig. 9.
Syndet is palmitoylated at the cysteine-rich
domain. Cells expressing syndet, delta-syndet, and syndet C1S/C3S
were labeled with [3H]palmitate for 2 h, as
described (19). Wild-type and mutated syndet proteins were
immunoprecipitated using the affinity purified anti-syndet antibody.
Immunoprecipitates were analyzed by SDS-PAGE electrophoresis and
fluorography (see "Experimental Procedures"). The gel was exposed
for 20 days. This experiment was repeated three times with similar
results.
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DISCUSSION |
Pairing of individual SNAREs before fusion may be at least one of
the mechanisms by which cells maintain different organelles as unique
compartments. In this light, it is essential that individual SNARE
components be targeted to their correct destination. In this paper, we
have identified the cysteine-rich region of the t-SNARE component,
syndet, as critical for subcellular localization of syndet. We find
that deletion of the cysteine-rich domain of syndet abolishes membrane
binding. This result is in agreement with the finding that a mutant
SNAP-25, lacking its cysteine-rich domain, is soluble (20). SNAP-25 is
palmitoylated in the central nervous system and in transfected cells
(18, 19). Syndet, syndet C1S/C3S, but not the mutant lacking the
cysteine-rich domain, are palmitoylated in the transfected AtT-20 cell
model. Thus, it is likely that fatty acylation is necessary for the
binding of syndet to membranes. Moreover, we find that the
cysteine-rich domain of syndet is essential for targeting the protein
to the plasma membrane. We conclude that the cysteine-rich domain of syndet has a dual role in membrane binding and plasma membrane targeting. Unlike the deletion of the cysteine-rich domain, deletions of 32 amino acids at the amino terminus domain or 25 amino acids at the
carboxyl terminus domain of syndet do not have an appreciable effect on
protein distribution.
Our data show that the distribution of syndet which has been mutated at
three cysteines, syndet C2S/C4S/C5S, is shifted from the plasma
membrane to a diffuse intracellular localization. We also find that
syndet C4S/C5S, C2S/C4S, and C2S/C5S are unable to accumulate at the
cell periphery and are found in the cytoplasm. Syndet C4S and syndet
C5S are both localized at the plasma membrane. Our observations
indicate that: 1) at least two cysteines within the -Cys2-
Cys4-Cys5- triplet must be substituted to
prevent syndet localization at the cell surface; 2) single and double
mutations of cysteines within the
-Cys2-Cys4-Cys5- triplet have the
same effect on syndet distribution, irrespective of which residue is
changed. We conclude that Cys2, Cys4, and
Cys5 are critical for plasma membrane localization and may
have similar and somewhat overlapping roles.
Doubly lipid modified peptides are stably anchored to membranes (30).
Since syndet and SNAP-25 have multiple cysteines, some cysteines may
target these proteins to intermediate localizations along the
biosynthetic route or to specialized domains at the plasma membrane.
Indeed, it appears that SNAP-25 A, SNAP-25 B, and SNAP-23 accumulate in
specific domains at the cell surface (5, 31-33). Modifications at
cysteines may also modulate their binding to membranes (19) or the
formation of SNARE complexes (34).
Because syndet Cys1 and Cys3 are not conserved
in SNAP-25 B and SNAP-25 A, it is reasonable to conclude that
modifications at these residues may account at least in part for
differences in the localization and function of these proteins. We have
generated a mutant syndet protein in which both cysteines are
substituted with serines. We find that, unlike the double mutants
within -Cys2-Cys4-Cys5- triplet, a
fraction of syndet C1S/C3S remains as an integral membrane component at
steady state. Consistent with the concept that acylation of syndet is
necessary for its binding to membranes, we find that syndet C1S/C3S is
palmitoylated. We conclude that Cys1 and Cys3,
unlike Cys2, Cys4, and Cys5, are
not absolutely required for membrane binding. We also find that
mutations at Cys1 and Cys3 of syndet result in
localization of the mutated protein to the plasma membrane, as well as
a Golgi-like compartment and the cytosol. Interestingly, a mutation of
a specific cysteine palmitoylation site in Src protein kinase
p56lck, can shift a fraction of the protein from the plasma
membrane to a Golgi compartment (35). Our results support the concept that the cysteine-rich domain of syndet plays a major role in subcellular localization of the protein. We conclude that the distribution of syndet, like that of Src protein kinase p56lck,
may be influenced by the configuration of the cysteines.
SNAP-25 associates with the Golgi compartment at the cell body of the
neurons and then undergoes fast axonal transport as an acylated protein
(18, 36). Thus, SNAP-25 reaches the plasma membrane by association with
transport vesicles. We find that syndet C1S/C3S is found in the
cytosol, the Golgi, and the plasma membrane. The distribution of
mutated syndet may mimic the steps of syndet or SNAP-25 biosynthetic
routes. If this is the case, then acylations within the
-Cys2-Cys4-Cys5- triplet may be
sufficient to bind syndet to a Golgi localization. Acylation(s) at
Cys1 and/or Cys3 may shift wild-type syndet
from the trans-Golgi network to the plasma membrane. In the absence of
Cys1 and Cys3 transit to the plasma membrane is
slow and mutated syndet C1S/C3S accumulates at the Golgi localization.
Alternatively, it is also possible for mutated syndet C1S/C3S to
localize both at the Golgi and at the plasma membrane directly, from
the cytosol. In mast cells, SNAP-23 relocates from the plasma membrane
to dense core granules in response to stimulation (37). Our results
indicate that modifications at the cysteine-rich motif can induce
redistribution of syndet to an intracellular compartment. However, in
AtT-20 cells, syndet C1S/C3S is redistributed to an intracellular
compartment different from dense core granules. More work is necessary
to understand whether modifications at the cysteine-rich motif play a
role in changes of SNAP-23/syndet or SNAP-25 cell distribution under
different conditions.
In conclusion, our paper shows that the cysteine-rich domain of syndet
is a major determinant for the subcellular localization of the protein.
We propose that modifications at the clustered cysteines may control
syndet distribution and function in cells.