From the Institute for Molecular Bioscience and the
Department of Physiology and Pharmacology, University of Queensland,
St. Lucia, Queensland 4072, the § Joint Protein Structure
Laboratory, Ludwig Institute of Cancer Research and the Walter and
Eliza Hall Institute, Parkville, Victoria, Australia 3052, the
¶ Welcome Trust Centre for Molecular Mechanisms in Disease,
University of Cambridge, Addenbrooke's Hospital, Cambridge CB22XY,
United Kingdom, and the
Department of Physiology and
Biophysics, University of Iowa, Iowa City, Iowa 52242
Received for publication, November 30, 2000, and in revised form, February 23, 2001
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ABSTRACT |
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Syntaxin 7 is a mammalian target soluble
N-ethylmaleimide-sensitive factor attachment protein
receptor (SNARE) involved in membrane transport between late endosomes
and lysosomes. The aim of the present study was to use immunoaffinity
techniques to identify proteins that interact with Syntaxin 7. We
reasoned that this would be facilitated by the use of cells producing
high levels of Syntaxin 7. Screening of a large number of tissues and
cell lines revealed that Syntaxin 7 is expressed at very high levels in
B16 melanoma cells. Moreover, the expression of Syntaxin 7 increased in
these cells as they underwent melanogenesis. From a large scale
Syntaxin 7 immunoprecipitation, we have identified six polypeptides
using a combination of electrospray mass spectrometry and
immunoblotting. These polypeptides corresponded to Syntaxin 7, Syntaxin
6, mouse Vps10p tail interactor 1b (mVti1b), In eukaryotic cells, proteins are transported between
intracellular organelles by a series of membrane transport steps. The ability of discrete organelles to fuse in a highly specific way is
central to all membrane-trafficking events and relies on a series of
molecular events. One event is the formation of a protein complex
between sets of molecules found within the transport vesicle (v-SNAREs)1 and the target
membrane (t-SNAREs). Much of the work that has led to the formulation
of this hypothesis has been performed in the mammalian synapse (1).
Here the R- or v-SNARE, VAMP2, forms a complex with two Q- or t-SNARE
proteins, Syntaxin 1a and SNAP25. This ternary complex consists of a
four- Sequencing projects are revealing the existence of an ever increasing
number of SNARE family members (6). In the yeast S. cerevisiae there are eight different Syntaxin-like proteins, and
in mammalian cells, at least 20 have been identified to date (7, 8). A
major challenge is to establish which transport steps each of these
proteins regulates. This will require establishing the intracellular
location of these proteins as well as their binding partners. Syntaxins
1, 2, 3, and 4 localize to the plasma membrane (9); Syntaxin 5 operates
between the endoplasmic reticulum and the Golgi apparatus; and
Syntaxins 6, 7, 8, 13, and 16 are found in the endosomal system of
mammalian cells (10-14).
The present study focuses on Syntaxin 7. Syntaxin 7 is localized to
compartments within the endosomal system where it regulates transport
between the late endosome and the lysosome (14-16). We have reported
previously that Syntaxin 7 interacts with at least one v-SNARE, VAMP8
(14). Recent analysis of VAMP8 has demonstrated a requirement for this
v-SNARE in the homotypic fusion of both early endosomes and late
endosomes (17). Another v-SNARE, VAMP7, has also been implicated in
late endosomal transport (16, 18), but its relationship to Syntaxin 7 has not yet been explored. This highlights the need for a more thorough
characterizaton of Syntaxin 7 binding partners. The aim of the present
study was to identify Syntaxin 7-binding proteins using a biochemical
approach. To this end, we took advantage of the fact that Syntaxin 7 is expressed at very high levels in the melanoma cell line, B16. Using B16
cells as a source of Syntaxin 7, we used an immunoprecipitation approach combined with mass spectrometry to identify mVti1b, Syntaxin 6, and Cells and Membrane Preparation--
B16 Melanoma cells were
kindly provided by Dr. Peter Parsons (QIMR, Brisbane, Australia) and
were cultured in RPMI medium (Life Technologies, Inc.) supplemented
with 10% fetal calf serum (Life Technologies, Inc.). For immunoblot
analysis, total membranes were prepared as follows. Cells were lysed by
passage through a 27-gauge needle in HES buffer (20 mM
Hepes, 0.5 mM EDTA, 250 mM sucrose) containing
protease inhibitors (10 µg/ml leupeptin, 10 µg/ml aprotinin, and
250 µM phenylmethylsulfonyl fluoride) before
centrifugation at 150,000 × g for 30 min. The membrane pellet was resuspended in HES buffer containing 1% Triton X-100 and
incubated on ice for 1 h, after which insoluble material was removed by centrifugation at 17,500 × g for 10 min.
Triton X-100-soluble extracts of rat tissues, obtained from male Wistar
rats, were prepared by homogenization of the relevant tissues in HES
buffer containing protease inhibitors. Homogenates were incubated for 30 min at 4 °C in the presence of 1% Triton X-100, after which insoluble material was removed by centrifugation at 17,500 × g for 10 min.
Antibodies, Plasmids, and Immunoblot Analysis--
Polyclonal
antiserum against mouse Syntaxin 7 was obtained by immunizing rabbits
with a chimeric protein consisting of the cytosolic domain of Syntaxin
7 fused to GST (pGST-Syn7a) (14). Antibodies raised against the
GST portion of the antigen were removed by passing the antiserum over a
column of GST linked to Affi-Gel (Bio-Rad). The flow-through from this
column was subsequently passed over a second column made by coupling
the GST-Syn7antigen to Affi-Gel. Syntaxin 7-specific antibodies were
eluted from this column with 100 mM glycine, 150 mM NaCl (pH 2.8). These affinity-purified antibodies were
then titrated to pH 7.5 using Tris-HCl (pH 8). The VAMP8 antibodies
have been described previously (14). Antibodies to VAMP7 were raised
against GST fusion protein containing the entire cytosolic portion of
VAMP7, which was expressed from pPL815. Similarly, antibodies against
Syntaxin 6 were raised by immunizing rabbits with a GST protein
containing the entire cytosolic tail of this protein (pGST-Syn6; 14) or
were purchased from Transduction Laboratories. A plasmid encoding the
cytosolic tail of human Syntaxin 13 fused to GST and an antibody
produced against this fusion protein were kindly provided by Dr. R. Teasdale (Institute for Molecular Bioscience, University of
Queensland). The EEA1 antibody was the generous gift of Dr. Ban Hock
Toh (Monash Medical School, University of Melbourne, Australia).
Antibodies against mVti1b (19) were kindly provided by Dr. G. Fischer
von Mollard (University of Gottingen, Germany). Antibodies against
cytosolic domains of Syntaxin 4 and VAMP3 have been described
previously (20). Membrane samples were subjected to SDS-PAGE and
subsequently transferred to polyvinylidene difluoride membranes.
Membranes were blocked in 1% (w/v) non-fat dried milk and then
incubated with primary antibodies in phosphate-buffered saline
containing 0.1% (v/v) Tween 20 and 1% (w/v) non-fat dried milk for
1 h at room temperature at dilutions that were optimized for each
antibody. Visualization of antibody-labeled bands was achieved with the
use of horseradish peroxidase-labeled secondary antibodies purchased
from Amersham Pharmacia Biotech, and Supersignal Dura chemiluminescent
substrate was from Pierce. Immunoblotting competition assays were
performed by including the relevant GST fusion protein antigen (100 µg/ml) during incubation with the primary antibody. The GFP-Syn6
plasmid that encodes the full-length murine Syntaxin 6 fused to the C
terminus of enhanced GFP (eGFP) was a kind gift from Dr. Jeffrey Pessin
(University of Iowa) (21).
Immunofluorescence Microscopy--
Cells were grown to 70%
confluence on glass coverslips and fixed in 2% paraformaldehyde for 30 min. In some cases B16 cells were transiently transfected with the
GFP-Syntaxin 6 vector using the LipofectAMINE reagent according to the
manufacturer's instructions (Life Technologies, Inc.). This plasmid
generated a protein of the expected molecular mass (~60 kDa)
which could be specifically immunoblotted with antibodies specific for
either Syntaxin 6 or GFP (data not shown). After fixation, cells were
quenched for 5 min in 150 mM glycine, washed in
phosphate-buffered saline, and permeabilized with 0.2% Triton X-100 in
phosphate-buffered saline for 15 min. After blocking coverslips with
2.5% normal swine serum for 30 min at room temperature primary
antibody incubations were performed in 1% normal swine serum in
phosphate-buffered saline for 1 h at room temperature, followed by
an appropriate fluorescein isothiocyanate- or Texas Red-labeled
secondary antibody (Molecular Probes, Eugene, OR) for 30 min at room
temperature. Cells were viewed using a 63×/1.4 Ziess oil immersion
objective on a Zeiss Axiovert fluorescence microscope, equipped with a
Bio-Rad MRC-600 laser confocal imaging system. Images were imported
into Adobe PhotoshopTM (Adobe Systems Inc.) and assembled as indicated.
Immunoprecipitation--
Affinity matrices were prepared by
first binding antibodies (affinity-purified anti-Syn7 antibodies with
or without an excess of GST-Syn7 fusion protein and purified rabbit IgG
antibodies) to protein A-Sepharose beads (Amersham Pharmacia Biotech).
Antibodies were covalently cross-linked to protein A beads using
dimethyl pimelimidate (Pierce). Triton X-100-soluble membrane
extracts prepared from B16 cells, which had been cultured for 7 days,
were prepared as outlined above and incubated with antibody-coated beads for 1 h in the presence of 150 mM NaCl, 20 mM Hepes, and 0.5 mM EDTA. The beads were
washed in the same buffer containing 1% Nonidet P-40. Elution of bound
proteins was achieved by boiling for 10 min in Laemmli sample buffer
containing 4% SDS. Proteins were separated on a 5-15%
SDS-polyacrylamide gradient gel and visualized by staining with either
silver or Coomassie Brilliant Blue. Individual protein bands were
excised and subjected to sequence analysis.
Protein Sequencing and Mass Spectrometer Analysis--
In-gel
proteolytic digestion (using 0.5 µg of trypsin) of resolved
proteins was performed as described previously (22). An electrospray
ionization ion trap mass spectrometer (LCQ Finnigan MAT, San Jose, CA)
coupled on-line with a capillary HPLC (Hewlett-Packard model 1090A)
modified for capillary chromatography was used for peptide sequencing.
The column used in this study was a 150 × 0.20-mm (inner
diameter) capillary column (Brownlee RP-300, 7 µm C8) manufactured
using a polyvinylidene difluoride end frit A 60-min linear gradient
(flow rate 1.7 µl/min) was used from 0-100% B, where solvent A was
0.1% v/v aqueous trifluoroacetic acid, and solvent B was 0.1% aqueous
trifluoroacetic acid in 60% acetonitrile. The electrospray ionization
parameters were as follows: spray voltage, 4.5 kV; sheath gas and
auxiliary gas flow rates, 5 and 30 (arbitrary value), respectively;
capillary temperature, 150 °C; capillary voltage, 20 V; and tube
lens offset, 16 V. The sheath liquid used was 2-methoxyethanol (99.9%
HPLC grade) delivered at a flow rate of 3 µl/min. The electron
multiplier was set to Specificity of the Syntaxin 7 Antibody--
A Syntaxin 7-specific
antibody was raised against a bacterial fusion protein containing the
cytosolic tail (residues 1-234) of Syntaxin 7 fused to GST. Antibodies
that specifically recognize the Syntaxin 7 portion of this fusion
protein were affinity purified as described under "Materials and
Methods." This antibody recognizes one band of molecular mass 40 kDa
(Fig. 1A, lane 1).
Immunolabeling of this band was completely inhibited in the presence of
an excess of the GST-Syn7 antigen (lane 2). In contrast, a
fusion protein consisting of the analogous region of Syntaxin 13 fused
to GST had no significant effect on the recognition of this band
(lane 3). In addition, the mobility of the polypeptide
recognized by the Syntaxin 7 antibody was significantly different from
the bands labeled with antibodies specific for either Syntaxin 13 or
Syntaxin 6 (Fig. 1A, lanes 4 and 5).
To validate further the specificity of the Syntaxin 7 antibody we
performed immunofluorescence microscopy using B16 melanoma cells. The
antibody labeled a tubulovesicular compartment that was concentrated in
the perinuclear region of the cell (Fig. 1B). This labeling
was not significantly different upon inclusion of excess GST-Syn13
fusion protein. However, in the presence of excess GST-Syn7 the
immunolabeling was almost completely absent, consistent with the
immunoblotting data (Fig. 1A). Collectively, these data show
that our Syntaxin 7 antibody is specific and does not recognize other
closely related Syntaxin isoforms.
Syntaxin 7 Is Expressed at High Levels in B16 Cells--
We have
shown previously that Syntaxin 7 is widely expressed in rodent tissues
and that it regulates a membrane transport step within the late
endosome/lysosomal system of mammalian cells (14). In view of this
function we reasoned that cell types that are specialized for late
endosomal/lysosomal biogenesis might up-regulate the machinery that is
involved in these transport steps. Melanocytes are a specialized cell
type whose primary function is the biogenesis of melanosomes, which
represent lysosome-like organelles (24). Consistent with this notion is
our finding that the levels of the Syntaxin 7 protein in the B16
melanosome cell line is ~10-fold higher than in all other tissues and
cell lines tested (Fig. 2). In contrast,
the level of Syntaxin 4, which localizes to the plasma membrane, was
much more uniform across all tissues and cell types, including B16
cells. A similar uniformity was observed for Syntaxin 6 (Fig. 2) and
Syntaxin 13 (data not shown) across these tissues and cell types.
Intriguingly, VAMP8, a v-SNARE that has also been implicated in late
endosomal transport (17), is also expressed at very high levels in B16
cells compared with other tissues and cell lines (Fig. 2).
Immunoprecipitation of Syntaxin 7-interacting Proteins--
The
high levels of Syntaxin 7 which we observed in B16 cells gave us a good
opportunity to find interacting proteins using a coimmunoprecipitation
approach. The Syntaxin 7-specific antibody (Fig. 1) was covalently
linked to protein A-Sepharose, forming an affinity matrix. This was
used to immunoprecipitate Syntaxin 7-containing protein complexes from
a B16 cell extract. Using a non-ionic detergent solubilized extract of
B16 cells we were able to immunoprecipitate Syntaxin 7 with high
efficiency and specificity (Fig. 3).
Initially, to identify proteins that interact with Syntaxin 7, immunoprecipitates were subjected to SDS-PAGE followed by silver
staining (Fig. 3B). We were able to detect a number of
proteins that coprecipitated with Syntaxin 7 under these experimental
conditions (see first and fourth lanes).
Most of these protein bands could not be detected when the
immunoprecipitation was performed using a control IgG (second
lane) or when the incubation with the Syntaxin 7 antibody was
performed in the presence of excess recombinant GST-Syn7 (fifth
lane). The most prominent bands detected were of average molecular
mass 230, 130, 120, 90, 35, and 30 kDa. To identify these proteins we
increased the scale of the immunoprecipitation procedure so that bands
could be visualized by staining with Coomassie Brilliant Blue (see Fig.
3C). These bands were excised, and tryptic peptide products
were sequenced using electrospray mass spectrometry. The sequences
obtained are shown in Table I. Several of
the bands yielded very clear sequence data and gave positive identities
when these sequences were used for BLAST searching of the protein data
base. The doublet at 130/120 kDa contained sequences corresponding to
the protein phosphatase 110-kDa regulatory subunit (25) and a tyrosine
kinase associated protein known as BAP-135 (26). In an effort to verify
the presence of BAP-135 in the Syntaxin 7 immunoprecipitate B16 cells
were incubated with pervanadate because this causes a marked
stimulation of BAP-135 tyrosine phosphorylation in B cells (26).
However, immunoblotting of the Syntaxin 7 immunoprecipitate, obtained
from these cells, using a phosphotyrosine antibody failed to reveal any
detectable product corresponding to BAP-135 (data not shown). Of
particular interest, we were able to detect a number of peptide sequences in the Syntaxin 7 immunoprecipitate corresponding to Syntaxin
7,
To verify the identity of these proteins as Syntaxin 7-interacting
partners we immunoblotted immunoprecipitates obtained from B16 cells
using antibodies specific for a variety of these proteins. As shown in
Fig. 4, Syntaxin 6, VAMP8, and mVti1b
were all enriched in the Syntaxin 7 immunoprecipitate to almost the
same extent as Syntaxin 7. None of these proteins was present when the
immunoprecipitation was performed using preimmune serum-coated protein
A beads (Fig. 4, second lane). The immunoprecipitation
efficiency of Syntaxin 7 was ~50% (Fig. 4, compare first
and third lanes). In the case of Syntaxin 6, VAMP8, and
mVti1b ~20%, 20, and 10% of each protein was coprecipitated with
Syntaxin 7, indicating that each of these proteins was highly enriched
in the Syntaxin 7 complex suggesting that they likely form stable SNARE
complexes in vivo. It has been reported recently that
another v-SNARE, VAMP7, can play a role in the delivery of epidermal
growth factor to a degradative compartment, most likely the late
endosomal/lysosomal system, where VAMP7 is localized (18). Although we
did not detect this protein in the Syntaxin 7 immunoprecipitate by
MS/MS we were able to detect coprecipitation of VAMP7 with Syntaxin 7 by immunoblotting (Fig. 4). VAMP7 was enriched in the Syntaxin 7 immunoprecipitate to the same extent as VAMP8 (Fig. 4). Although a
significant proportion of VAMP7 was found in the Syntaxin 7 immunoprecipitate, our inability to detect it by MS/MS raised the
possibility that it may be expressed at lower abundance than other
proteins. We also immunoblotted these fractions with antibodies
specific for Syntaxin 4 and VAMP3 to investigate the specificity of the
immunoprecipitation. Fig. 4 shows that neither Syntaxin 4 nor VAMP3 was
found in the Syntaxin 7 immunoprecipitate to any significant extent.
Although we did detect a faint band for both proteins in the Syntaxin 7 immunoprecipitate upon overexposure, this represents a very minor
enrichment (~1%) compared with other SNARE proteins (see above).
Immunolocalization of Syntaxin 7 and VAMP8 in B16 Cells--
The
identification of Syntaxin 6 as a Syntaxin 7-binding partner was
somewhat surprising because this t-SNARE has been immunolocalized previously to the trans-Golgi network in PC12 cells (13).
However, it was clear from the immunoblotting data (Fig. 4) that a
significant amount of Syntaxin 6 coprecipitates with Syntaxin 7 in B16
cells. Hence, one interpretation of these data is that Syntaxin 6 participates in Syntaxin 7-mediated fusion events as a t-SNARE light
chain. To verify further that Syntaxin 7 and Syntaxin 6 might form a complex in vivo we conducted a series of double labeling
immunolocalization experiments (Fig. 5).
First, consistent with our previous data (14) there was very little
overlap between Syntaxin 7 and the early endosomal protein EEA1 (Fig.
5, A and B). Syntaxin 7 was localized to large vesicles
scattered throughout the cytoplasm and clustered in the perinuclear
region of the cell. Based on our previous studies these likely
correspond to late endosomes (14). We next compared the distribution of
GFP-Syn6 with mVti1b, Syntaxin 7, and Syntaxin 13 in B16 cells. As
shown in Fig. 5, E and F, there was substantial
overlap between Syntaxin 6 and Syntaxin 7, particularly in the
dispersed vesicles. There was an additional pool of Syntaxin 6 in the
perinuclear region, which did not appear to contain Syntaxin 7. This
pool presumably corresponds to the trans-Golgi network (13).
Similarly, there was also a high degree of overlap between mVti1b and
Syntaxin 6 in cytosolic vesicles. In contrast, we observed very little
if any overlap between Syntaxin 6 and Syntaxin 13. Intriguingly, the
distribution of Syntaxin 13 was also quite distinct from EEA1 in these
cells being enriched in a very fine vesicular tubular network (Fig. 5G).
Expression of Different SNARE Proteins during
Melanogenesis--
Our earlier finding that production of Syntaxin 7 is up-regulated upon induction of melanogenesis raised the possibility
that this expression would be part of a developmental program that would drive the coordinate expression of other proteins that
functionally interact with Syntaxin 7. Thus, to determine if Syntaxin 7 expression correlated with the expression of the proteins we identified
as interacting with Syntaxin 7, we examined lysates prepared from B16
cells at different stages of melanosome development. The synthesis and
storage of melanin in intracellular organelles can be observed readily
as an increased deposition of dense black melanin granules or
melanosomes that are scattered throughout the cytoplasm of the cell. As
is shown in Fig. 6, a lysate prepared
from B16 cells at different stages of melanogenesis demonstrated an
increased deposition of melanin with increased time in culture. From
this time course we observed a parallel increase in the levels of
Syntaxin 7, Syntaxin 13, and the v-SNAREs, VAMP8 and VAMP7. In
contrast, the levels of Syntaxin 4 and Syntaxin 6 remained constant
throughout this time course (Fig. 6). We did note an increase in the
expression of two other v-SNARE proteins, VAMP2 and VAMP3. However, the
time course of these changes did not parallel the increase in melanin production. Because of our experience with different sublines of B16
cells behaving slightly differently, we also analyzed this phenomenon
in a B16 cell line that only underwent melanogenesis after stimulation
by The conventional view is that melanosomes represent a specialized
type of lysosome (24). As such, melanosomes are likely to
be derivatives of the late endosomal pathway. Previously it has been
found that the induction of melanogenesis results in the coordinate
expression of tyrosinase and lysosome-associated membrane protein 1 (29), implying that these proteins are expressed as part of a
developmental program that facilitates the biogenesis of melanosomes.
Consistent with this idea we have found that expression of Syntaxin 7 is also induced during melanogenesis in B16 cells (Fig. 6). Thus, it is
likely that elevated levels of Syntaxin 7 facilitate melanogenesis
given the established role of Syntaxin 7 in lysosomal biogenesis (14,
16). We also find that the levels of the v-SNARE proteins that interact
with Syntaxin 7 are also increased upon melanocyte differentiation,
suggesting that the biochemical interactions we observe between these
proteins have significance in vivo.
Among the cohort of proteins we found associated with Syntaxin 7 were
the Q-SNAREs, mVti1b and Syntaxin 6, and the R-SNARE, VAMP7 (Fig. 3).
Furthermore, we also found VAMP8 associated with Syntaxin 7 in B16
cells, consistent with our previous findings in Madin-Darby canine
kidney cells (14). The identification of these novel Syntaxin 7-binding
partners will permit a more detailed analysis of the composition of the
various SNARE complexes that control fusion events within the late
endocytic pathway. These interactions are best interpreted in light of
recent experiments from Rothman and colleagues (3, 4, 30). The core
SNARE complex comprises a four-helix bundle, and this may be formed by
either three or four different proteins. One helix is contributed by
the v- or R-SNARE, which resides in the "vesicle" membrane, and the
remaining three helices are contributed by the t- or Q-SNAREs, residing
in the "target" membrane. Thus, Syntaxin 7 is predicted to regulate
vesicle transport events in the late endosomal system in tandem with
other Q-SNAREs.
Our studies strongly suggest that mVti1b is at least one of the
relevant Q-SNARE partners for Syntaxin 7. Based on both sequence homology and functional studies in yeast, both mVti1a and mVti1b appear
to be orthologs of the yeast Vti1p (31). Yeast Vti1p is best
characterized as a generic Q-SNARE because it interacts with the
endoplasmic reticulum to Golgi t-SNARE, Sed5p, with the endosomal
t-SNAREs, Pep12p, Tlg1p, and Tlg2p; and with the vacuolar t-SNARE,
Vam3p (5, 32-34). The identification of mVti1p as a Syntaxin
7-interacting protein is consistent with the notion that Syntaxin 7 is
functionally equivalent to Vam3p in yeast (14, 15) because Vam3p and
Vti1p form a functional SNARE complex to regulate vacuolar transport.
Thus, these results bolster the concept that the molecular regulation
of vacuolar fusion and lysosomal fusion is highly conserved between
these distinct organisms.
Consistent with previous results (14), our studies substantiate an
interaction between VAMP8 and Syntaxin 7. In addition, antibodies
against VAMP8 that block its ability to participate in SNARE core
complex formation block homotypic fusion of both early and late
endosomes in vitro (17). These data, combined with
observations that homotypic fusion between late endosomes is blocked by
reagents designed to disrupt Syntaxin 7 function, suggest that Syntaxin
7 and VAMP8 are the relevant Q- and R-SNAREs for homotypic fusion
reactions in the late endosomal pathway. However, we also find that
Syntaxin 7 is in a complex with VAMP7. The structural and experimental
models to date indicate that the three-helix Q-SNARE subcomplex
interacts with a single v-(R)-SNARE to complete the four-helix bundle
core complex (2-4, 30). Based on these experiments it is
unlikely that one SNARE complex will contain two different R-SNARE
helices. This raises the possibility that Syntaxin 7 can form two
distinct SNARE complexes, one with VAMP7 and one with VAMP8. Whether a
Syntaxin 7·VAMP8 complex catalyzes fusion events that are distinct
from those catalyzed by a Syntaxin 7·VAMP7 complex remains to be
definitively determined. Like VAMP8, VAMP7 has been localized to late
endosomal compartments in some cell types and is implicated in the
control of fusion events within the late endocytic pathway (16, 18).
Thus, these separate complexes may control fusion of late endosomes
with endosomes derived from different origins. Alternatively, a
Syntaxin 7·VAMP8·mVti1b complex may regulate homotypic fusion of
late endosomes, whereas a Sytnaxin 7·VAMP7·mVti1b complex may
facilitate fusion of late endosomes with lysosomes. Interestingly, our
VAMP8 antibody has no detectable effect in an in vitro late
endosome/lysosome fusion assay derived from rat liver fractions (14),
whereas VAMP7 antibodies are inhibitory in this
assay.3 To distinguish
between these possibilities it will be important to discern carefully
which type of fusion events are under investigation in these types
of assay.
Based on previous structural studies and on the interaction of Syntaxin
7 with two different v-(R)-SNAREs and the Q-SNARE Vti1p we would
predict that at least one other protein would be required in this
complex to complete the four-helix bundle requisite within a core
complex (2-3, 30). Although this could be accomplished by the presence
of two mVti1b subunits, we were surprised to find that in B16 cells,
the most likely protein to fulfill this role is Syntaxin 6. Syntaxin 6 was originally described as a Golgi t-SNARE that could interact with
the v-SNAREs, cellubrevin and VAMP2 (13). However, more recently it has
been shown that Syntaxin 6 interacts with other SNAREs including SNAP23
(36), Syntaxin 13, and the Q-SNARE Syntaxin
16.4 What emerges from these
data is the view that within a particular specialized cell type, SNARE
proteins may be recruited selectively to new locations to fulfill
distinct and selective functions. One particularly informative example
is the observation that in neutrophils, Syntaxin 6 is targeted to the
plasma membrane where it mediates regulated secretion (36).
Furthermore, VAMP7 also appears to be localized differentially in
specialized cell types such as neurons, polarized epithelial cells, and
basophils (37-39). Thus, the localization of Syntaxin 6 to late
endosomal compartments in B16 cells is consistent with this view (Fig.
5). These observations indicate that the specialized nature of
different cells may dictate the steady-state localization of SNARE
proteins, thus highlighting the potential limitation in using members
of the SNARE family as generic compartment-specific markers.
Furthermore, these observations require that understanding the
compartmentation and function of the endocytic system in terms of
particular SNARE proteins must be undertaken on a cell-by-cell basis.
The significance of the association between Syntaxin 7 and PP1M
(a particular regulatory subunit of protein phosphatase I) remains an
area for future investigation. Given the abundance and specificity of
this protein in our immunoprecipitates it seems likely that this
protein indeed associates with Syntaxin 7 in vivo. Because
Syntaxin 7 mediates lysosomal fusion, a possible role for PP1M could be
in the fusion process. Interestingly, in vitro homotypic
fusion of yeast vacuoles requires a protein phosphatase I that is
activated by calcium and calmodulin (40). Although a functional role
has not yet been demonstrated for a protein phosphatase I in lysosomal
fusion, there is a requirement for calcium and calmodulin in lysosomal
fusion which mirrors the requirement for calcium and calmodulin in
homotypic vacuolar fusion (41). Intriguingly, melanosomes are
transported to the cell periphery in a manner that is dependent on
myosin Va (42) and Rab27a (43). Mouse mutations (dilute and
ashen, respectively) in these proteins result in coat color
abnormalities. Furthermore, changes in the phosphorylation state of
myosin V are correlated with its activity (44, 45). Thus PP1M could
regulate the activity of myosin V and via its interaction with Syntaxin
7, or PP1M could regulate interaction between the melanosome and myosin
V. Interestingly, movement of pigment granules in angelfish
melanophores is also blocked by inhibitors of protein phosphatase I
(35).
-synaptosome-associated protein (SNAP), vesicle-associated membrane protein (VAMP)8, VAMP7, and
the protein phosphatase 1M regulatory subunit. We also observed partial
colocalization between Syntaxin 6 and Syntaxin 7, between Syntaxin 6 and mVti1b, but not between Syntaxin 6 and the early endosomal t-SNARE
Syntaxin 13. Based on these and data reported previously, we propose
that Syntaxin 7/mVti1b/Syntaxin 6 may form discrete SNARE complexes
with either VAMP7 or VAMP8 to regulate fusion events within the late
endosomal pathway and that these events may play a critical role in melanogenesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helical bundle containing one helix from both Syntaxin 1a and
VAMP2 with the remaining two helices being contributed by SNAP25 (2).
SNARE complexes that regulate traffic to the cell surface in both
mammalian and yeast cells contain three distinct proteins, whereas most
intracellular SNARE complexes appear to be comprised of four separate
proteins: one v-SNARE and three t-SNAREs (3, 4). For example, in
Saccharomyces cerevisiae endoplasmic reticulum to
Golgi transport is regulated by the
Sed5p·Bos1p·Sec22p·Bet1p complex, whereas vacuolar
transport is regulated by a complex comprising
Vam3p·Vam7p·Vti1p·Nyv1p (3). The Syntaxin isoform, or
t-SNARE heavy chain (3), associated with each complex appears to be
highly specific to a particular vesicle transport step, whereas the
light chain t-SNAREs associate with multiple complexes. Vti1p,
for example, interacts with Syntaxin homologs involved in
Golgi/endosome, intra-Golgi, vacuolar, and prevacuolar transport steps
(5).
-SNAP as Syntaxin 7-binding proteins. In addition, we find
that Syntaxin 7 forms a complex with both VAMP7 and VAMP8, suggesting
that the t-SNARE Syntaxin 7 may regulate distinct fusion events within
the endocytic pathway by associating with distinct subsets of partner
SNARE proteins.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
860 V. In both MS and MS/MS modes, the trap was
allowed a maximum injection time of up to 200 ms. The automatic gain
control parameter was turned on for all experiments, ensuring that the
number of ions in the trap was automatically kept to a constant preset
value. The range scanned in MS mode was 350-2,000 kDa, and in MS/MS
the range varied according to the mass of the ion selected for MS/MS. After acquiring one scan in MS, the most intense ion in that spectrum above a threshold of 1 × 105 was isolated for
subsequent zoom scan (to determine charge state), then
collision-induced dissociation or MS/MS in the following scans. The
dissociation energy for MS/MS was set to 55%. All spectra were
recorded in centroid mode. The sequences of individual peptides were
identified using the SEQUEST algorithm (incorporated into the Finnigan
Xcalibur-Biomass software) (23). Spectra not identified by SEQUEST
were interpreted manually using de novo methods.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (46K):
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Fig. 1.
Specificity of the Syntaxin 7 antibody. A rabbit polyclonal antibody was raised against a GST
fusion protein containing the cytosolic tail of Syntaxin 7. The
antibody was affinity purified and used either for immunoblotting
(panel A) or immunofluorescence microscopy (B).
Equal amounts of protein (10 µg) from a B16 mouse melanoma extract
were subjected to SDS-PAGE followed by immunoblotting using either the
Syntaxin 7 antibody (lanes 1-3) or antibodies against
Syntaxin 13 (lane 4) or Syntaxin 6 (lane 5).
Immunoblotting using the Syntaxin 7 antibody was also performed in the
absence or presence of either excess GST-Syn7 protein (100 µg/ml,
lane 2) or excess GST-Syn13 protein (100 µg/ml, lane
3). The relative position of molecular weight markers is shown at
the right. In panel B, B16 cells were plated on
glass coverslips, fixed, permeabilized, and immunolabeled with the
Syntaxin 7 antibody alone or in the presence of either excess
GST-Syntaxin 7 (100 µg/ml) or excess GST-Syntaxin 13 (100 µg/ml).
Specific labeling was visualized using a Texas Red-conjugated secondary
antibody.
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[in a new window]
Fig. 2.
Expression of different SNAREs among
rat tissues and cell lines. Rat tissues were prepared as described
under "Materials and Methods" to obtain a Triton X-100-soluble
total membrane extract. Extracts from either Chinese hamster ovary
(CHO) or B16 cells were similarly obtained. Equal amounts of
lysate protein from each sample (10 µg) were subjected to SDS-PAGE
and immunoblotting with antibodies specific for the various SNARE
proteins as indicated. Each antibody immunolabeled one major band of
the appropriate molecular mass.
-SNAP (27), mVti-1b (GenBank NP058080), and VAMP8 (28). The
t-SNARE, Syntaxin 6, was also identified in the same band that
contained
-SNAP, migrating with an average molecular mass of 32 kDa.
The abundant band at 55 kDa corresponded to IgG. None of the other
bands that were identified using this approach yielded any detectable
peptide sequences.
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Fig. 3.
Immunoprecipitation of Syntaxin 7-containing
polypeptide complexes. Panels A and B,
affinity-purified Syntaxin 7 antibodies or an IgG control antibody was
covalently linked to protein A-Sepharose beads and incubated with a
detergent-solubilized B16 cell lysate in the absence or presence of
excess GST-Syn7 protein. In some cases the cell lysate was omitted from
the incubation to determine which bands were arising from beads alone.
Where indicated in panel B, 500 µg of B16 lysate was
included in each incubation. Samples were then subjected to SDS-PAGE
followed by either immunoblotting using the Syntaxin 7 antibody
(panel A) or silver staining (panel B) to
visualize protein complexes. The relative position of molecular mass
standards in the gel, which were run in parallel, is indicated at the
left in panel B. Panel C, to identify
some of the proteins indicated in the first lane of
panel B, the incubation of B16 extract with Syntaxin 7 antibodies was scaled up such that 400 µl of antibody-coupled beads
was incubated with 4 mg of lysate protein. Samples were subjected to
SDS-PAGE followed by Coomassie Blue staining. The bands indicated by
the arrows were excised from the gel, digested with trypsin,
and sequenced by electrospray ionization MS. Bands that gave positive
identifications are shown at the right of the gel, and the
corresponding peptide sequences are shown in Table I. It is noteworthy
that the VAMP8 band, although not visible on this reproduction, was
evident in gels freshly stained with Coomassie Blue.
Peptide sequences predicted from mass spectrometer analysis
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Fig. 4.
Detection of SNARE proteins in the Syntaxin 7 complex by immunoblotting. B16 lysates were incubated with protein
A beads that had been precoated with Syntaxin 7 antibodies in the
absence ( Syn7) or presence of excess
recombinant GST-Syn7 (control). These samples together with
an aliquot of B16 extract (1/10 of the starting material used for the
immunoprecipitation) were immunoblotted with antibodies specific for
Syntaxin 7, Syntaxin 6, mVti1b, VAMP3, VAMP8, VAMP7, and Syntaxin
4.
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Fig. 5.
Immunolocalization of Syntaxin 7 and VAMP8 in
B16 cells. B16 cells, cultured on glass coverslips, were double
labeled with antibodies specific for Syntaxin 7 and EEA1 (panels
A and B). B16 cells were also transiently transfected
with pEGFP-Syn6. At 24 h after transfection, cells were fixed,
permeabilized, and immunolabeled. Panels C and D
show GFP-Syn6 and anti-Syntaxin 7 labeling, panels E and
F show GFP-Syn6 and anti-mVti1b labeling, and panels
G and H show GFP-Syn6 and Syntaxin 13 labeling. Images
were obtained using a confocal immunofluorescence microscope.
-melanocyte-stimulating hormone. Similar results were obtained in
that Syntaxin 7, VAMP7, and VAMP8 were profoundly increased, whereas
levels of Syntaxin 6 and Syntaxin 4 were
unchanged.2
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Fig. 6.
Increased expression of various SNARE
proteins during B16 cell differentiation. B16 cells were
maintained as confluent monolayers for different times in culture
between 2 and 14 days. Cells were prepared as described under
"Materials and Methods" to obtain a Triton X-100-soluble total
membrane extract. Equal amounts of cell extract (10 µg of protein)
were subjected to SDS-PAGE followed by immunoblotting with antibodies
specific for proteins as shown. The increase in melanogenesis with time
in culture is indicated in the top panel by the progressive
darkening of the cell extracts.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Fiona Simpson, Rohan Teasdale, and Rob Parton for comments and assistance during the course of these studies. Many thanks also to Gabi Fischer von Mollard, Rohan Teasdale, Jeffrey Pessin, and Ban Hoch Toh for providing antibodies.
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FOOTNOTES |
---|
* This work was supported by the Human Frontiers Science Program (to D. E. J., R. C. P., and J. P. L.), by the Australian Research Council of Australia, and by American Heart Association Grant 9730275N. The Institute for Molecular Bioscience is a special research center of the Australian Research Council.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.
** National Health and Medical Research Council of Australia principal research fellow. To whom correspondence should be addressed. Tel. and Fax: 61-7-3365-4986; E-mail: D.James@imb.uq.edu.au.
Published, JBC Papers in Press, March 1, 2001, DOI 10.1074/jbc.M010838200
2 S. Richardson and R. C. Piper., unpublished data.
3 B. Mullock and J. P. Luzio, unpublished data.
4 S. Martin and D. James, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: SNARE(s), soluble N-ethylmaleimide-sensitive factor attachment protein receptor(s); t-, target; v-, vesicle; VAMP, vesicle-associated membrane protein; SNAP, soluble N-ethylmaleimide-sensitive factor attachment protein; SNAP25, synaptosomal-associated protein of 25 kDa; Sed5, suppressor of the erd2 deletion mutant; Bos1, Bet one suppressor; Sec, secretion; Bet, blocked early in transport; Vam, vacuolar morphology; Vti, Vps10p tail interactor; Nyv, new yeast v-SNARE; GST, glutathione S-transferase; Syn, Syntaxin; EEA1, early endosomal antigen 1; PAGE, polyacrylamide gel electrophoresis; GFP, green fluorescent protein; eGFP, enhanced green fluorescent protein; HPLC, high performance liquid chromatography; MS, mass spectrometry; MS/MS, tandem mass spectrometry; Tlg, t-SNARE of the late Golgi; Pep, peptidase-deficient gene; mVti1, mouse-Vti1.
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