Syntaxin 12, a Member of the Syntaxin Family Localized to the
Endosome*
Bor Luen
Tang
,
Andrew E. H.
Tan
,
Lay Kheng
Lim,
San San
Lee,
Delphine Y. H.
Low, and
Wanjin
Hong§
From the Membrane Biology Laboratory, Institute of Molecular and
Cell Biology, Singapore 117609, Republic of Singapore
 |
ABSTRACT |
We have cloned a new member of the syntaxin
family of proteins. The open reading frame encodes a polypeptide of 272 amino acids with potential coiled-coil domains and a C-terminal
hydrophobic tail. Northern blot analysis showed that the transcript is
fairly ubiquitous. A soluble recombinant form of the polypeptide
without the hydrophobic region binds to
-SNAP (soluble
N-ethylmaleimide-sensitive factor attachment protein) and
syndet/SNAP-23 in vitro. Polyclonal antibody raised against
the recombinant protein recognized a 39-kDa protein in the membrane
fraction of cell lysates. Indirect immunofluorescence studies using the
polyclonal antibody showed that the protein is localized to
intracellular membrane structures. Selective permeabilization studies
with digitonin and saponin indicate that the epitope(s) recognized by
the antibody is expose to the cytoplasm, consistent with the predicted
orientation characteristic of SNAP receptor molecules. Morphological
alterations of the staining pattern of the protein with brefeldin A and
wortmannin treatment indicate that the protein is localize to the
endosome. The cDNA we have cloned apparently corresponded to three
previously described expressed sequence tags named as syntaxins 12, 13, and 14, respectively. We therefore propose to retain the name syntaxin
12 for this protein.
 |
INTRODUCTION |
A biochemical and biophysical understanding of vesicular transport
at the molecular level has been facilitated by in vitro assays, which reconstitute transport processes in cell-free systems (1). Thus, molecular components required for both vesicle budding (2)
and vesicle docking/fusion processes (3-4) have been isolated. The
N-ethylmaleimide-sensitive factor
(NSF),1 an ATPase whose
activity regulates the formation and dissociation of fusion complexes,
is the first cytosolic factor characterized as such. NSF works in
conjunction with another soluble factor, the soluble NSF attachment
protein (SNAP) in mediating vesicle docking (1). Membrane components
that are responsible for determining the specificity of the docking and
fusion of the right vesicles to the right membranes are eventually
identified based on the ability to interact with SNAP (4). These are
known as SNAP receptors (SNARE). SNAREs can be broadly divided into two
classes. Those present on transport vesicles are the v-SNAREs, and
those present on the target membranes are the t-SNAREs. Thus, the SNARE hypothesis, a working hypothesis proposed by Rothman and co-workers (3,
4), holds that a transport vesicle chooses its target for fusion when
v-SNAREs pair with the cognate t-SNAREs at the target membrane. Genetic
dissections of the yeast secretory pathway and the biochemical
characterization of molecules involved in synaptic vesicle docking and
fusion have resulted in the isolation and/or molecular cloning of
putative SNARE molecules that are structurally related (5-6). However,
isolation of SNAREs in the constitutive secretory and endocytotic
pathway in mammalian cells remains difficult. This difficulty has been
overcome in part by the availability of an expanding data base of
expressed sequence tags (EST) and efficient data base search and
sequence alignment programs (7).
The first member of the syntaxin family of proteins, syntaxin 1A, was
first characterized as a neuronal-specific protein involved in the
regulation of neurotransmitter release (8). Its localization to the
plasma membrane and its interaction with the synaptic vesicle v-SNARE
synaptobrevin point to its function as a t-SNARE. Subsequently, a
family of syntaxin-related molecules that shares 23-84% amino acid
identity has been identified (6, 9). These syntaxins are more
ubiquitous in their expressions in various tissues, which is indicative
of their possible functions in other vesicular transport steps in the
cell. These syntaxins also display a variety of cellular localizations
within the secretory pathway. Whereas syntaxins 2, 3, and 4 are
apparently cell surface proteins (6, 10-11), syntaxin 5 and syntaxin 6 are localized to the Golgi region (6, 9).
The transport from the early endosome to the late endosome and lysosome
is a major route of intracellular membrane trafficking. However, little
is known at the molecular level about the mechanisms regulating
membrane interactions in the endocytic pathway beyond early endosomes.
Recently, it has been shown that COP1 coat components participate in
endosomal transport (12). Using an in vitro transport assay
to study the biochemical properties of endosome docking and fusion
events, Robinson et al. (13) have shown that NSF and SNAPs
are required for several steps of endosomal membrane transport. Of the
known SNAREs, only the v-SNARE cellubrevin has been shown to have an
endosomal localization (14). Should the mechanism of vesicular
transport in the endosomes not differ too drastically from its exocytic
counterpart, one might expect to find members of the syntaxin family
functioning as t-SNAREs in the endosomal membranes. In this report, we
present such a molecule.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Cell lines were primarily from the American Type
Culture Collection. Monoclonal antibody against trans-Golgi network 38 (TGN38) was kindly provided by Dr George Banting (University of
Bristol, United Kingdom). Expressed sequence tag clones were generated by the Washington University MERCK EST project and were obtained from
the IMAGE consortium. Syndet cDNA (15) was kindly provided by Dr.
G. Baldini (Columbia University, New York). Syntaxin 1A cDNA (6)
was kindly provided by Dr. R. Scheller (Stanford University, CA).
mSEC13 cDNA (16) was kindly provided by Dr. Anand Swaroop (University of Michigan).
Methods--
Data base searches were performed with the various
BLAST algorithms available at the National Center for Biotechnology
(NCBI) World Wide Web server. Library screening, cloning, and DNA
sequencing were performed using standard methods as described (17).
Northern blot analysis was performed using a rat multiple tissue
Northern blot from CLONTECH.
In vitro translation of various constructs was performed
using in vitro translation kits from Promega according to
the manufacturer's protocol. For in vitro binding assays
(18), the cytoplasmic domain of syntaxin 12 (amino acids 1-248) was
generated by the polymerase chain reaction and cloned into the the
plasmid pBSK (Stratagene). [35S]Methionine-labeled
translation product was incubated with glutathione-Sepharose beads
coated with either glutathione S-transferase (GST),
GST-
SNAP, or GST-syndet in binding buffer (20 mM Hepes,
pH 7.5, 25 mM NaCl, 3% glycerol, 7 mM
MgCl2, 1 mM CaCl2, and 1 mM EDTA) with 0.1% bovine serum albumin and 0.5% Nonidet
P40 at 4 °C for 3 h. The beads were washed twice with the
complete incubation buffer, twice in buffer without bovine serum
albumin, and twice in buffer without bovine serum albumin and Nonidet
P-40. SDS sample buffer was then added, and the SDS eluates were
analyzed by SDS-polyacrylamide gel electrophoresis.
The cytoplasmic domain of the protein is expressed either as
hexahistidine-tagged or GST fusion proteins in bacteria. The fusion
proteins were also used to immunize rabbits. Polyclonal antibodies were
affinity-purified from serum harvested after several booster injections
by the fusion proteins immobilized on nitrocellulose strips.
Cells were maintained in RPMI medium supplemented with 10% fetal
bovine serum. Immunofluorescence microscopy was performed as described
previously (19-20). Cells plated on coverslips and subjected to
various treatments were fixed with 3% paraformaldehyde followed by
sequential incubation with the primary antibodies and fluorescein
isothiocyanate or rhodamine-conjugated secondary antibodies.
Fluorescence labeling was visualized using an Axiophot microscope (Carl
Zeiss, Inc., Thornwood, NY) with epifluorescence optics or MRC600
(Bio-Rad) confocal laser optics.
 |
RESULTS |
Molecular Cloning and Sequencing of a Novel Member of the Syntaxin
Family--
Data base searches have allowed us to identified human
ESTs (accession numbers R21569 and N99549) potentially coding for a
syntaxin-like molecule. A complete cDNA was isolated from a rat
brain cDNA library, and sequencing revealed a 272-amino acid open
reading frame as shown in Fig.
1A. The predicted amino acid
sequence has a stretch of 22 hydrophobic residues at the C terminus, as
illustrated by a Kyte-Doolittle hydrophobicity plot (Fig.
1B). This primary structure is characteristic of a hydrophobic tail anchor. The polypeptide has several potential regions
that may form coiled-coil structures, as revealed by the Coils version
2.1 program (Fig. 1C).

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Fig. 1.
Molecular cloning of a novel rat syntaxin.
A, the DNA sequence and derived amino acid sequence of the
coding region of rat syntaxin 12. The putative transmembrane is
boxed. The coiled-coil region, which is most homologous to
other known syntaxins, is underlined. B,
Kyte-Doolittle hydrophobicity plot of the primary sequence of syntaxin
12 performed by the DNA Strider 1.1 program. C, coils output
for syntaxin 12 coils 2.1 analysis of potential coiled-coil domains of
syntaxin 12. The window of analysis is 21 amino acids wide.
|
|
A data base search using the NCBI BLAST program revealed that the
coding sequence has the highest homology with members of the syntaxin
family, particularly in the coiled-coil region preceding the C-terminal
transmembrane domain (Fig.
2A). A multiple tissue Northern blot with the full-length cDNA showed that the transcript has a fairly ubiquitous expression, being more abundant in brain, lung,
and kidney (Fig. 2B).

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Fig. 2.
A, the alignment of the coiled-coil
region of rat syntaxin 12 with rat syntaxins 3 and 4 (MegAlign program
of DNASTAR). Identical residues are shaded. B,
Northern blot analysis of syntaxin 12. H, heart;
B, brain; S, spleen; L, lung;
Li, liver; SM, skeletal muscle; K,
kidney; T, testis; kb, kilobase pairs.
C, schematic diagram showing how the translated sequence of
the human ESTs AA167677, R29508, and T08774 match with the coding
region of rat syntaxin 12 (syn12). The translated sequence
of the human ESTs were based on BLAST search results returned by the
server of the National Center for Biotechnology Information, using the TBLASTN program to search the dbest data base.
|
|
Based on ESTs identified by data base searches, a series of 10 novel
syntaxins has been previously described (21). A search of the
GenBankTM dbest data base revealed several human ESTs that,
in view of their sequence homology, represent the human homologs of our
rat cDNA. Among these, three human ESTs (R29508, AA167677, and T08774) have been listed as syntaxin 12, syntaxin 13, and syntaxin 14, respectively (21). Fig. 2C is a schematic diagram of how the
translated sequence of these ESTs match with the coding region of the
rat cDNA. As shown, the limited and inaccurate sequence information
of these ESTs only allowed each of them to be matched with a portion of
the coding region. There is a short overlap between AA167677 and
R29508. T08774 matches to the C-terminal portion of the rat protein.
Apparently, these assumed syntaxins 12, 13, and 14 represent the human
homolog of the rat protein. Based on the limited sequence data alone,
these ESTs were assumed to represent individual syntaxin-like
molecules. Our results therefore caution against the assignment of
individual identity to an EST before full-length sequence information
of each is available. To avoid any further confusion in the
nomenclature of new members of the syntaxin family, we have retained
the name syntaxin 12 for this protein.
Syntaxin 12 Is a SNAP Receptor Molecule Localized to the
Endosome--
The predicted primary structure of syntaxin 12 and its
homology to other syntaxins suggest that it is a SNARE molecule. We sought to confirm this by investigating if syntaxin 12 binds to
-SNAP in vitro. [35S]Methionine-labeled
translation product of the soluble cytoplasmic domain of both syntaxin
1A and syntaxin 12 was incubated with glutathione-Sepharose beads
coated with either GST or GST-
SNAP. As shown in Fig.
3, the binding of the syntaxin 1A
cytodomain to GST-
SNAP is significantly higher than to GST itself.
Such is also the case for syntaxin 12. On the other hand, mSEC13 (20), a protein with multiple
-transducin or WD-40 repeats known to participate in protein-protein interactions, did not exhibit
significant binding to GST-
SNAP. The ability of syntaxin 12 to bind
SNAP is in good agreement with its putative function as a SNARE.

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Fig. 3.
Binding of syntaxin 12 to -SNAP and
syndet/SNAP23 in vitro. The cytoplasmic domain of
syntaxin 1A (syn 1A) and syntaxin 12 (syn12) and
the full-length cDNA of mSEC13 were translated and labeled in
vitro with [35S]methionine. Binding was performed
with glutathione beads coated with GST (indicated by ) and either
GST- -SNAP or GST-SNAP23 (indicated by +) as described under
"Experimental Procedures." Molecular size markings on the left are
in kDa.
|
|
Syntaxin 1 is known to exist in complex with another neuronal-specific
SNARE molecule, SNAP-25 (22-23). A novel nonneuronal molecule that
binds syntaxin and synaptobrevin and is homologous to SNAP-25 has since
been cloned from human B lymphocyte (18), and based on its molecular
size, is called SNAP-23. Recently, syndet, a ubiquitous mouse protein
homologous to SNAP-25 has also been cloned (15). Based on the sequence
similarity, syndet appear to be the mouse homolog of human SNAP-23. We
sought to determine if syntaxin 12 could also interact in
vitro with syndet/SNAP-23. As shown in Fig. 3, both syntaxin 1A
and syntaxin 12 binds to syndet/SNAP-23 with high specificity, whereas
mSEC13 again exhibits no binding. The ability of syntaxin 12 to bind
SNAP-23 is highly suggestive of its functional similarity with other
members of the syntaxin family.
That syntaxin 12 is indeed a membrane-anchored protein was confirmed by
the fact that its full-length translated product could not be stripped
off membranes by high salt (1 M KCl) or high pH (sodium
bicarbonate, pH 11) treatment but could be solubilized with a detergent
such as Triton X-100 (Fig.
4A).To further characterize the molecule, rabbit polyclonal antibodies were raised using
bacterially expressed fusion protein. The affinity-purified antibody
detected a ~39-kDa band by immunoblot analysis of the in
vitro translated product of the full-length cDNA and NRK cell
lysates (Fig. 4B). Detection can be abolished by co- or
preincubation of the antibody with excess amount of the fusion protein
(not shown).

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Fig. 4.
A, syntaxin 12 is an integral membrane
protein. The full-length cDNA of syntaxin 12 was translated in the
presence of canine pancreatic microsomes. The translation mix was
either untreated, treated with 1 M KCl, 0.1 M
sodium bicarbonate, pH 11, or 1% Triton X-100 for 1 h on ice. An
aliquot was then loaded onto a 15% sucrose cushion and centrifuged at
100,000 × g for 45 min. The pellet (P) and
supernatant (S) of each treatment were loaded on adjacent lanes. The lane marker contains molecular size markers with
their indicated size in kDa. B, Western immunoblot analysis
of syntaxin 12 cold translation mix (lane 1), total NRK cell
lysate (lane 2), 100,000 × g NRK cell
lysate supernatant (lane 3), and 100,000 × g NRK cell lysate pellet (lane 4). Molecular size
markings on the left are in kDa.
|
|
As a first step toward functional characterization of syntaxin 12, we
performed indirect immunofluorescence microscopy to localized the
protein in several cell lines. As shown in Fig. 5, the antibody-stained intracellular
structures clustered at the perinuclear region of mouse (MEF), human
(HeLa), and rat (NRK) cells. To confirm the membrane topology of
syntaxin 12, cells were permeabilized with 20 µg/ml digitonin, which
would selectively permeabilized the plasma membrane while leaving the
internal membranes intact, or 1 mg/ml saponin, which would permeabilize
all membranes. Cells were then double-labeled with syntaxin 12 polyclonal antibody and a monoclonal antibody against the lumenal
domain of the Golgi protein mannosidase II. As shown in Fig. 5,
digitonin-permeabilized cells were labeled for syntaxin 12 only and not
mannosidase II. Permeabilization of the plasma membrane was indeed
achieved because under the same conditions, cytoplasmic tubulin was
also labeled (not shown). As expected, both syntaxin 12 and mannosidase
II were labeled in saponin-permeabilized cells. Syntaxin 12 is thus membrane-anchored, with a large portion of its N terminus exposed in
the cytoplasm, a topology characteristic of all syntaxins but not all
SNAREs identified to date.

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Fig. 5.
Indirect immunofluorescence analysis of
syntaxin 12. Cells were fixed with 4% paraformaldehyde and
incubated with affinity-purified rabbit polyclonal antibody against rat
syntaxin 12 (syn12) alone (top panel, mouse
embryonic fibroblast (MEF) and HeLa cell lines) or together
with a monoclonal antibody against rat mannosidase II
(manII) (bottom panel, NRK cells). This is
followed by incubation with fluorescein isothiocyanate-labeled
anti-rabbit IgG alone (top panel) or together with
TRITC-labeled anti-mouse IgG (bottom panel).
Permeabilization of cells in the lower panel is described in
text. Bar, 10 µm.
|
|
All syntaxins discovered to date were localized to either the Golgi
region or the cell surface, indicating that they function along the
exocytic pathway. As shown in Fig. 6, the
antibody labeled perinuclear structures in NRK cells. The labeling
pattern in NRK cells resembles that of the the TGN marker, TGN38 (24),
double-labeled in the same cell although not completely colocalized.
The fungal metabolite brefeldin A (BFA) has varying effects on the
morphology of subcellular organelles and on the distribution of various
markers on these organelles (25). The distribution of Golgi markers to
the endoplasmic reticulum (25) and the collapse of TGN markers TGN38
(26) and furin proprotein convertase (27) and endosomal markers
(28-30) into the microtubule-organizing center upon BFA treatment had
been extensively documented. The effect of BFA on the morphology of a
particular protein is therefore often useful in determining its
subcellular localization. Treatment of cells with 10 µg/ml BFA
resulted in the collapse of the structure into a compact structure
characteristic of the microtubule-organizing center, colocalizing well
with that of TGN38. This result suggests that the perinuclear staining
of syntaxin 12 is not that of the Golgi apparatus (which under this
condition would have redistributed to the endoplasmic reticulum) but
may be that of the TGN or the endosomes. BFA, however, also causes a
fusion of the endosomes with the trans-Golgi network (28), and
endosomal markers behave quite like TGN markers at the end point of the
BFA effect.

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Fig. 6.
Subcellular localization of rat syntaxin
12. Cells, either untreated (C) or treated with 10 µg/ml BFA or 1 µM wortmannin (wort) for
1 h were fixed with 4% paraformaldehyde and incubated with
affinity-purified rabbit polyclonal antibody against rat syntaxin 12 (syn 12) and a monoclonal antibody against rat TGN38. This
is followed by incubation with fluorescein isothiocyanate-labeled anti-rabbit IgG and TRITC-labeled anti-mouse IgG before being processed
for indirect immunofluorescence and confocal microscopy. Bar, 10 µm.
|
|
To further determine the exact localization of the rat syntaxin and
differentiate between the two possibilities, we treat cells with
wortmannin, the phosphatidylinositol 3-kinase inhibitor (31). This drug
has been shown to alter the morphology of endosomes but not the Golgi
apparatus or the trans-Golgi network (32-33). Although wortmannin
treatment did not alter the perinuclear Golgi staining marked by the
TGN38 monoclonal antibody, the perinuclear structure marked by the rat
syntaxin antibody in the same cells were converted into swollen
vacuoles, characteristic of wortmannin-induced changes to the stainings
of endosomal markers such as the mannose 6-phosphate receptor (32). We
observed this as well in another cell line, L2 (not shown). The above
results strongly suggest that the rat syntaxin-like molecule is
localized to a BFA and wortmannin-sensitive endosomal compartment.
 |
DISCUSSION |
We have therefore cloned a novel member of the syntaxin family
with a unique subcellular localization. None of the syntaxins 1-6
published to date are associated with the endosome. Exogenous expression of syntaxin 1A does result in intracellular localization in
Madin-Darby canine kidney cells, and exogenous expression of syntaxin 3 does have an intracellular component (10). These intracellular
stainings were, however, shown not to be endosomal in nature but rather
colocalized with a lysosomal marker (10). Another novel syntaxin, known
as syntaxin 7, has recently been cloned in our laboratory (34) as well
as by Wang et al. (35). Based on its homology to yeast and
plant vacuolar syntaxins, Wang et al. proposed that syntaxin
7 may have a role in trafficking between the Golgi apparatus and the
lysosomes. Our immunolocalization data, however, suggest that syntaxin
7 is localized to the endosomes (34). In view of the localizations of
syntaxin 7 and syntaxin 12, the transport machinery of the endocytic
pathway, like its exocytic counterpart, also utilizes members of the
syntaxin family.
What may the function of syntaxin 12 be? There are several
possibilities. Judging by its compact, perinuclear staining, syntaxin 12 may well reside in a late endosomal compartment. Indeed it does not
colocalize with transiently internalized transferin (not shown).
However, we could not rule out that small amounts of syntaxin 12 may
reside in the early endosomes. If solely localize to a late endosomal
compartment, syntaxin 12 may function to receive vesicles either from
the TGN or the early endosome or participate in the recycling of
surface receptors. Elucidation of its exact role in transport awaits
experiments involving effective functional disruption either by the
introduction of negative dominant mutants, inhibitory antibodies, or
targeted disruption of the gene.
 |
ACKNOWLEDGEMENTS |
We thank Dr. George Banting for monoclonal
antibody against TGN38, Dr. G. Baldini for syndet cDNA, Dr. R. Scheller for syntaxin 1A cDNA, Dr. Anand Swaroop for mSEC13
cDNA, Dr. S. H. Wong for GST-
SNAP, and Mr. Robin Philps for
protein sequencing.
 |
FOOTNOTES |
*
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF035632.
The first two authors contributed equally to this work.
§
Supported by a grant from the Institute of Molecular and Cell
Biology. To whom correspondence should be addressed: Membrane Biology
Laboratory, Institute of Molecular and Cell Biology, 30 Medical Dr.,
Singapore 117609, Republic of Singapore. Tel.: 65-874-3762; Fax:
65-779-1117; E-mail: mcbhwj{at}leonis.nus.sg.
1
The abbreviations used are: NSF,
N-ethylmaleimide-sensitive factor; RPMI medium, Rosewell Park Memorial
Institute medium; SNAP, soluble NSF attachment proteins, SNARE, SNAP
receptor; NRK, normal rat kidney; EST, expressed sequence tags; GST,
glutathione S-transferase; TGN, trans-Golgi network; BFA,
brefeldin A.
 |
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