1 Membrane Traffic and Neuronal Plasticity, INSERM U536, Institut du
Fer-à-Moulin, 75005 Paris, France
2 Morphogenesis and Cell Signaling, CNRS UMR 144, Institut Curie, 75005 Paris,
France
* Author for correspondence (e-mail: galli{at}idf.inserm.fr)
Accepted 10 March 2003
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Summary |
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Key words: Membrane fusion, SNARE proteins, TI-VAMP, Cellubrevin, Syntaxin
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Introduction |
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Membrane trafficking is a highly dynamic process. The molecular machinery
mediating membrane fusion is continuously cycling between distinct
intracellular compartments. This is especially true for v-SNAREs. For example,
exocytic SNAREs located on vesicles that have fused with the plasma membrane
need to be rapidly internalized from the cell surface in order to participate
in other rounds of fusion. Moreover, one v-SNARE might participate in several
fusion steps, as illustrated by cellubrevin, which mediates exocytosis and
endosome-to-TGN trafficking (Galli et al.,
1994; Mallard et al.,
2002
). Similarly, the yeast v-SNAREs Snc1 and Snc2 are involved in
exocytosis and in retrograde transport to endosomes and to the Golgi complex
(Gurunathan et al., 2000
;
Paumet et al., 2001
).
Despite this continuous trafficking, SNAREs are localized at steady state
in specific intracellular compartments, and the specificity of membrane fusion
is ensured. Identifying the mechanism responsible for compartmental
specificity is a major issue in this field. The emerging picture is that there
are complementary layers of regulation that are essential for the
establishment and maintenance of organelle compartmentalization in eukaryotic
cells. Among these regulators there are members of the Rab GTPase family and
their effectors (Pfeffer.,
2001; Siniossoglou and Pelham,
2001
), tethering factors
(Shorter et al., 2002
) and the
SNAREs themselves (McNew et al.,
2000
; Scales et al.,
2000
). Studies using an in vitro fusion assay suggested that
compartmental specificity could be achieved to a large extent by the inherent
specificity of cognate interactions between SNAREs
(McNew et al., 2000
).
In this study, we sought to test the hypothesis that the steady state
subcellular localization and destination of v-SNAREs, and therefore the
specificity of membrane fusion, depend on the ability of v-SNAREs to interact
with their cognate t-SNAREs and thus would ultimately depend on the
localization of t-SNAREs. If this was true, interfering with the targeting of
a t-SNARE should in turn affect the distribution of its specific v-SNARE
partners without altering the localization of its non-cognate SNAREs. To test
this hypothesis, we took advantage of the fact that expression of the plasma
membrane t-SNARE syntaxin 1 in non-neuronal cells leads to the accumulation of
this protein in the Golgi apparatus (at short time points after transfection),
followed by its redistribution to the ER (after longer times of transfection).
Whereas co-transfection of syntaxin 1 and nSec1/Munc18-1 restores normal
plasma membrane targeting of syntaxin 1
(Perez-Branguli et al., 2002;
Rowe et al., 2001
;
Rowe et al., 1999
). Our data
show that ectopic expression of syntaxin 1 in the ER redirected its cognate
v-SNAREs without affecting non-cognate ones. Therefore our results support the
proposal that the localization of a v-SNARE may be linked to the distribution
of its cognate t-SNAREs and provide in vivo evidence for SNARE-mediated
specificity of membrane fusion.
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Materials and Methods |
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The human cDNA of endobrevin, originally cloned from CaCo2 cells, was
obtained from ATCC (EST176564) (Paumet et
al., 2000). The N-terminal GFP fusion protein GFP-TIVAMP has been
described previously (Martinez-Arca et
al., 2000
). For the N-terminal GFP fusion proteins GFP-cellubrevin
(GFP-Cb) and GFP-endobrevin (GFP-Eb) the cDNAs of cellubrevin
(Galli et al., 1998
) and
endobrevin were cloned into the pEGFP-C3 vector (Clontech, Palo Alto, CA). For
the C-terminal GFP-fusion constructs TIVAMP-GFP and Cb-GFP, we generated a
superecliptic variant of the ecliptic pHLuorin (G. Miesenbock, Sloan Kettering
Memorial Hospital, NY) containing two mutations (F64L and S65T) that lead to
enhanced fluorescence (Sankaranarayanan
and Ryan, 2000
). The cDNAs of rat syntaxin 1A and nSec1 have been
described previously (Bennett et al.,
1992
; Garcia et al.,
1994
). The cDNA of rat syntaxin 7 was from R. Jahn [Max-Planck
Institute, Göttingen, FRG (Antonin et
al., 2000
)].
Cell culture and transfection
HeLa cells were cultured and transfected as described previously
(Martinez-Arca et al., 2000)
or with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the
manufacturer's instructions. MDCK cells were grown in DMEM with 10% FCS.
Stable MDCK clones expressing GFP-TIVAMP, GFP-Cb or GFP-Eb were produced by
electroporation and selected with G418 (0.4 mg/ml). MDCK clones were
transfected with Lipofectamine 2000. For the nocodazole treatment MDCK cells
plated the day before were treated with 5 µM nocodazole for 1 hour at
37°C before transfection. The drug was left in the medium until fixation
of the cells.
Immunoprecipitation
HeLa cells were lysed 24 hours after transfection in TSE (50 mM Tris pH
8.0, 10 mM EDTA, 150 mM NaCl) plus 1% Triton X-100 with protease inhibitors
for 1 hour at 4°C under continuous shaking. The supernatant resulting from
centrifugation at 20,000 g for 30 minutes, adjusted to a
protein concentration of 1 mg/ml, immunoprecipitated at 4°C overnight,
followed by the addition of 50 µl of magnetic beads (Dynabeads, Dynal,
Compiègne, France). After 3 hours at 4°C the magnetic beads were
washed four times with TSE plus 1% Triton X-100 and eluted with sample buffer.
Proteins were resolved by SDS-PAGE and detected by western blotting with the
ECL system (SuperSignal West Pico Chemiluminescent Substrate, Pierce,
Rockford, IL).
Antibody uptake and transferrin recycling
HeLa cells transfected with Cb-GFP or TIVAMP-GFP plus syntaxin 1 were
incubated in the presence of 5 µg/ml anti-GFP monoclonal antibody in
culture medium for 1 hour at 37°C, washed extensively with PBS, fixed with
4% PFA and processed for immunofluorescence. For the transferrin recycling
assay, cells were starved for 30 minutes in DMEM/15 mM HEPES pH 7.5, incubated
in the same medium with biotinylated human transferrin (SIGMA, St Louis, MI)
for 1 hour at 37°C, washed extensively and either fixed immediately
(transferrin uptake) or left at 37°C for 1 hour before fixation
(transferrin release). For the detection of the CD63 molecules at the plasma
membrane, cells were incubated for 1 hour at 4°C with the monoclonal
anti-CD63 AD1 antibody (dilution 1/50) in DMEM/15 mM HEPES pH 7.5, washed in
PBS and fixed.
Immunocytochemistry and confocal microscopy
Cells were fixed with 4% PFA and processed for immunofluorescence as
described previously (Coco et al.,
1999). For the immunofluorescence with anti-SNAP-23 and with
anti-Na+/K+ ATPase, cells were fixed/permeabilized with
cold methanol as described elsewhere
(Faigle et al., 2000
).
Secondary antibodies (Molecular Probes, Eugene, OR) were coupled to Cy3 and
Alexa 488 for double labeling and to Cy3 and Cy5 for triple labeling with
GFP-fused proteins. Confocal laser scanning microscopy was performed using a
SP2 confocal microscope (Leica, Mannheim, FRG). Images were acquired by
sequential excitation with 488 nm, 543 nm and 633 nm laser beams. Low
magnification images were obtained using a 63x lens (zoom 2-3), the
image size being set to 1024x1024 pixels. The high magnification images
were obtained using a 100x lens (zoom 10-12), the image size being set
to 256x256 pixels. Images were assembled using Adobe Photoshop (Adobe
Systems, San Jose, CA).
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Results |
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This was, in fact, the case. In cells co-transfected with syntaxin 1 and nSec1, endogenous TI-VAMP displayed a typical vesicular pattern, the same as in non-transfected cells, whereas in cells transfected with syntaxin 1 alone the vesicular pattern was changed to a diffuse reticular staining reminiscent of the ER (Fig. 1B). In the same way, endogenous cellubrevin displayed a vesicular pattern with a strong concentration in the perinuclear region in non-transfected cells or in cells co-transfected with syntaxin 1 and nSec1, whereas in syntaxin-1-expressing cells cellubrevin was widespread throughout the cytoplasm, and its perinuclear enrichment was lost (Fig. 1C and data not shown). Notably, this drastic change in distribution upon syntaxin 1 expression in HeLa cells was not seen for non-cognate v-SNAREs of syntaxin 1, such as endobrevin (Fig. 1C).
A detailed study of the compartment to which endogenous TI-VAMP and
cellubrevin were redistributed in syntaxin-1-transfected HeLa cells was
hampered both proteins being expressed at only a low level, and it is only
their normal localization to vesicular structures that facilitates their
detection. However, when the vesicular distribution was lost in
syntaxin-1-expressing cells, the pattern of both v-SNAREs became diffuse, as
expected for membrane proteins redistributing from discrete punctate
structures to a much larger surface such as the ER. Therefore, to further
analyze whether or not TI-VAMP and cellubrevin colocalized with syntaxin 1 in
the ER, we used stable MDCK cell lines expressing GFP-tagged versions of
TI-VAMP, cellubrevin and endobrevin. Expression of syntaxin 1 in MDCK cells
also results in its intracellular retention, whereas co-transfection with
nSec1 restores its delivery to the surface
(Rowe et al., 2001). As shown
in Fig. 2, retention of
syntaxin 1 in the ER of MDCK cells induced a drastic alteration in the
distribution of GFP-TIVAMP and GFP-cellubrevin (GFP-Cb) compared with
neighboring cells that did not express syntaxin 1. Moreover, triple labeling
with calreticulin showed triple colocalization of syntaxin 1 and GFP-TIVAMP or
GFP-Cb with calreticulin (light pink in the merge panels of
Fig. 2). In contrast,
GFP-endobrevin (GFP-Eb) was not affected. In the case of GFP-TIVAMP, vesicular
staining was completely replaced by a reticular pattern (compare the inset in
Fig. 2, which shows a cell
expressing syntaxin 1 displaying GFP-TIVAMP reticular distribution, with a
non-transfected cell showing GFP-TIVAMP vesicles). In the case of GFP-Cb, the
reticular pattern induced by expression of syntaxin 1 co-existed with some
residual vesicular staining. Interestingly, in these conditions the GFP-Cb
vesicles appeared perfectly aligned with the ER network whereas GFP-Eb
vesicles did not (compare insets for GFP-Cb and for GFP-Eb in
Fig. 2).
|
The redistribution of TI-VAMP and cellubrevin was not the result of
syntaxin 1 overexpression but rather of the retention of syntaxin 1 in the ER,
because cells co-transfected with syntaxin 1 and nSec1 expressed comparable
amounts of syntaxin 1 (Fig. 1B, Fig. 3B), and yet, in this
case, TI-VAMP and cellubrevin had a normal localization
(Fig. 1B and not shown). We
also analyzed the effect of overexpressing the endosomal syntaxin 7, which
forms, together with syntaxin 8 and Vti1b, an endosomal SNARE complex with
endobrevin (Antonin et al.,
2000) or TI-VAMP (Bogdanovic et
al., 2002
; Wade et al.,
2001
). However, in MDCK cells, exogenous syntaxin 7 was
distributed to endosomes, as previously reported for the endogenous protein
(Mullock et al., 2000
) and
colocalizes partially with TI-VAMP and endobrevin but not with cellubrevin
(Supplementary Fig. 1 available at
jcs.biologists.org/supplemental).
The lack of mislocalization of syntaxin 7 upon overexpression, which is most
probably caused by MDCK cells endogenously expressing this SNARE and the
machinery for its correct localization and function (including its putative SM
protein) did not allow further study of its role in cognate v-SNARE
distribution.
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Cognate v-SNAREs are retained in the ER of syntaxin-1-expressing
cells through their specific interaction with syntaxin 1
The mislocalization of TI-VAMP and cellubrevin in syntaxin-1-expressing
cells suggested that both v-SNAREs might be retained in the ER through a
direct and specific interaction with syntaxin 1. Therefore, we searched for
syntaxin-1-containing SNARE complexes. SNAREs are able to form promiscuous
interactions in vitro (Fasshauer et al.,
1999; Yang et al.,
1999
), although this is not the case in vivo or in vitro in
conditions where SNAREs are inserted in a membrane rather than in solution
(McNew et al., 2000
;
Parlati et al., 2000
;
Scales et al., 2000
).
Nevertheless, we first sought to clarify this point and verified that in our
experimental conditions SNARE complexes did not form in the detergent extract
of transfected HeLa cells. To test this, HeLa cells were either co-transfected
with GFP-Cb plus syntaxin 1 or transfected with each construct alone. Lysates
from co-transfected cells and a 1:1 mixture of lysates from cells expressing
each protein alone were immunoprecipitated with antibodies against GFP or
syntaxin 1. As shown in Fig.
3A, we could only detect co-immunoprecipitation of GFP-Cb and
syntaxin 1 when the cells were co-transfected, indicating that the complexes
were formed inside the cell and not during the extraction procedure.
We then obtained cells expressing either syntaxin 1 alone (retaining
syntaxin 1 at the ER, Fig. 1)
or together with nSec1 (expressing syntaxin 1 normally at the plasma membrane,
see Fig. 1B) and searched for
endogenous SNAREs co-immunoprecipitating with syntaxin 1. nSec1 was
efficiently expressed by the co-transfected cells but it did not
co-immunoprecipitate with anti-syntaxin 1 antibodies owing to the instability
of the nSec1syntaxin 1 complex
(Garcia et al., 1995). As
expected, SNAREs that do not normally interact with syntaxin 1, such as VAMP4,
endobrevin and Vti1b, were not found in the anti-syntaxin 1 immunoprecipitates
under any condition (Fig. 3B).
By contrast, the plasma membrane SNARE synaptosomal associated protein of 23
kDa (SNAP-23) was efficiently recovered, supporting the specificity of the
interactions. As expected, SNAP-23 co-immunoprecipitated with syntaxin 1 when
syntaxin 1 was correctly sorted to the plasma membrane, although to a lesser
extent than when syntaxin 1 was retained in the ER. By contrast, TI-VAMP and
cellubrevin could only be detected in anti-syntaxin 1 immunoprecipitates when
this t-SNARE was mistargeted to the ER. This is most probably because when
syntaxin 1 is co-expressed with nSec1 the latter negatively regulates the
availability of the former to participate in ternary SNARE complexes
(Perez-Branguli et al., 2002
;
Yang et al., 2000
). To detect
co-immunoprecipitation of cognate v-SNAREs, and because SNARE complexes are
short-lived (Peng and Gallwitz,
2002
), it would be necessary to pre-treat the cells with
N-ethylmaleimide (NEM) (Galli et al.,
1998
).
Mislocalization of syntaxin 1 induces the relocalization of its
cognate light chain SNAP-23
SNARE complex formation is controlled both by the strict specificity of the
recognition between v- and t-SNAREs and by the correct topology of this
interaction: namely one v-SNARE in one membrane and a t-SNARE complex formed
by one syntaxin and two light chains in the other membrane
(Parlati et al., 2000). Our
biochemical data regarding SNAP-23 suggested that when syntaxin 1 is retained
in the ER, the membrane of this compartment fulfilled the requirements to be a
target membrane for TI-VAMP and cellubrevin vesicles (i.e. the presence of
syntaxin 1 and the two light chains provided by SNAP-23). To confirm this
point, we analyzed the intracellular distribution of SNAP-23 in
syntaxin-1-expressing cells. As shown in
Fig. 4A, in non-transfected
cells or in cells co-transfected with syntaxin 1 plus nSec1, SNAP-23 displayed
the expected cell surface staining (arrows,
Fig. 4A) and the intracellular
vimentin-associated labeling previously described
(Faigle et al., 2000
) (data
not shown); by contrast, in cells expressing syntaxin 1 this plasma membrane
pattern was completely lost, and a significant pool of SNAP-23 colocalized
with syntaxin 1 (arrowhead, Fig.
4A). As a control, we analyzed the distribution of the plasma
membrane protein Na+/K+ ATPase, which does not interact
with SNAREs. As expected, the surface localization of
Na+/K+ ATPase was not affected by the expression of
syntaxin 1 (Fig. 4B).
Altogether, these data suggest that the ectopic expression of syntaxin 1 in
the ER induced the mislocalization of its cognate light chain by a direct
interaction.
Transport to the plasma membrane of TI-VAMP and cellubrevin is
impaired in syntaxin-1-expressing cells
The mislocalization of endogenous TI-VAMP and cellubrevin upon ectopic
expression of syntaxin 1, together with their specific interaction with
syntaxin 1 in the ER, suggested that under these conditions both proteins
might not be able to reach the cell surface. To further analyze this point, we
designed GFP fusion proteins of TI-VAMP and cellubrevin with the GFP tag fused
to the C-terminus, so that we could monitor their appearance at the plasma
membrane and endocytosis by incubating living cells with antibodies directed
against GFP and measuring antibody uptake. Cells transfected with TIVAMP-GFP
or Cb-GFP alone displayed typical plasma membrane and vesicular staining
patterns and efficiently bound the anti-GFP antibody in the culture medium
(Fig. 5, upper panels),
suggesting that TIVAMP-GFP and Cb-GFP reached the plasma membrane, as is the
case for the endogenous cellubrevin (Galli
et al., 1994). In contrast, when TIVAMP-GFP or Cb-GFP was
co-transfected with syntaxin 1, both proteins were retained in the ER and
failed to bind to the extracellular anti-GFP antibody
(Fig. 5, lower panels),
suggesting that transport to the plasma membrane and endocytosis was
abolished.
|
However, it is important to note that expression of syntaxin 1 did not induce pleiotropic effects on intracellular trafficking pathways because we have found that transferrin uptake and release were not affected in HeLa cells expressing either syntaxin 1 or syntaxin 1 plus nSec1 (Fig. 6), confirming the specificity of this experimental model.
Intracellular redistribution of TI-VAMP in syntaxin-1-expressing
cells is microtubule dependent
The appearance of TI-VAMP and cellubrevin in the ER of
syntaxin-1-expressing cells could be due either to redistribution from their
normal intracellular compartment or to sequestration of the newly synthesized
molecules. The latter possibility is unlikely because it would mean that the
half-lives of TI-VAMP and cellubrevin are short enough to allow detection of
only the newly synthesized pool after short times (8 hours) of syntaxin 1
transfection, and this is not the case (S.M.-A., V.P.-G., P.A., D.L. et al.,
unpublished). On the other hand, if the first hypothesis is true, inhibition
of vesicle-mediated translocation on microtubules should prevent the
redistribution of syntaxin 1 cognate v-SNAREs to the ER because the treatment
with nocodazole abolishes microtubule-dependent endosomal movement
(Matteoni and Kreis, 1987).
Thus, if relocalization of v-SNAREs to the ER in our syntaxin 1 overexpression
assay was the result of redistribution from endosomes, then nocodazole
treatment should inhibit it. To directly test this point we have used the
clone of MDCK cells that stably expresses GFP-TIVAMP. Treatment of these cells
with 5 µM nocodazole for 1 hour completely disrupted the microtubules, as
seen by staining with an anti-
-tubulin antibody
(Fig. 7A). As expected, the
GFP-TIVAMP pattern was slightly modified upon nocodazole treatment, the
GFP-TIVAMP-positive vesicles being more scattered throughout the cytoplasm and
less concentrated at the perinuclear area, probably because of an alteration
in the microtubule-organizing center. Interestingly, when the cells were
transfected with syntaxin 1 after the microtubules were disrupted by a 1 hour
pre-treatment with nocodazole, GFP-TIVAMP redistribution to the ER was
abolished (Fig. 7B) despite the
retention of syntaxin 1 in this compartment. By contrast, in cells that were
not treated with nocodazole, GFP-TIVAMP was mistargeted to the ER of
syntaxin-1-expressing cells only 8 hours after transfection
(Fig. 7B). These results
indicate that the relocalization of TI-VAMP to the ER in syntaxin-1-expressing
cells was not simply because of sequestration of newly synthesized molecules
but resulted from the redistribution of TI-VAMP from a pre-existing endosomal
compartment. These data also imply that TI-VAMP vesicles are able to move
bi-directionally along microtubules, in agreement with our observations by
time-lapse videomicroscopy in GFP-TIVAMP-transfected HeLa cells (S.M.-A. and
T.G., unpublished).
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Expression of syntaxin 1 in MDCK cells redirects TI-VAMP vesicles to
the ER
The results obtained after the nocodazole treatment suggested that upon
expression of syntaxin 1, TI-VAMP-containing vesicles fuse with the ER
membrane. If this is true, then we would expect that the cargo of these
vesicles would also be mislocalized in syntaxin-1-expressing cells. We have
previously found that TI-VAMP defines a new endosomal compartment in neurons
and PC12 cells and colocalizes with the tetraspanin protein CD63
(Berditchevski, 2001;
Coco et al., 1999
). We found
the same result in HeLa cells, as shown by the extensive colocalization of
TI-VAMP and CD63 (Fig. 8A,
upper panels). Therefore, we investigated the fate of CD63 molecules in cells
expressing and retaining syntaxin 1 in the ER. As shown in
Fig. 8A (lower panels), the
classic vesicular pattern of CD63 was partially lost in cells transfected with
syntaxin 1. In these conditions, CD63-positive vesicles were less abundant,
more heterogenous in size and shape and, importantly, the nuclear envelope and
fine reticular structures characteristic of the ER were labeled with the
anti-CD63 antibody (arrow in Fig.
8A). Furthermore, high magnification confocal images showed
partial colocalization of CD63 with the syntaxin 1 retained in the ER (inset
in Fig. 8A). This change in
distribution is not the result of a block in CD63 export from the ER, because
the same result was obtained when cells expressing syntaxin 1 were incubated
with cycloheximide for 3 hours prior to fixation (data not shown).
Furthermore, we have checked that CD63 does not colocalize with calreticulin
in untransfected cells or cells expressing syntaxin 1 plus nSec1 (S.M.-A.,
V.P.G., P.A., D.L. et al., unpublished). In normal conditions, the tetraspanin
protein CD63 cycles through the cell surface
(Kobayashi et al., 2000
).
Therefore, to confirm the effect of the ER retention of syntaxin 1 on CD63
distribution, we performed an antibody binding assay at 4°C to detect CD63
molecules at the plasma membrane. As expected, in non-transfected cells or in
cells transfected with syntaxin 1 plus nSec1, the anti-CD63 antibody was
efficiently bound, as a result of the presence of CD63 at the cell surface
(Fig. 8B, upper panels).
Strikingly, in cells expressing syntaxin 1 there was no detectable binding of
the antibody (Fig. 8B, lower
panels), supporting the redistribution of CD63 in these conditions. Moreover,
when the anti-CD63 antibody was incubated with living cells at 37°C for 90
minutes, it was efficiently internalized in non-transfected cells or in cells
transfected with syntaxin 1 plus nSec1, whereas no internalized antibody was
detected in cells transfected with syntaxin 1 alone (S.M.-A., V.P.G., P.A.,
D.L. et al., unpublished).
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Discussion |
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The redistribution of TI-VAMP and cellubrevin to the ER in fibroblasts
expressing syntaxin 1 points to a direct relationship between SNARE complex
formation and v-SNARE distribution and destination for the following reasons.
(1) It was due to syntaxin 1 mistargeting and not simply to syntaxin 1
overexpression because syntaxin 1 was expressed at the same level in the
presence and absence of nSec1 and yet no effect on cognate v-SNARE
distribution was seen in cells co-transfected with syntaxin 1 and nSec1. (2)
It was not the result of the retention of the newly synthesized v-SNARE
proteins but of an active microtubule-dependent redistribution of the
pre-existing v-SNARE molecules. (3) It recapitulated the specificity found in
v-/t-SNARE interactions because only syntaxin 1 cognate v-SNAREs were affected
by its mistargeting, and we detected a bona fide interaction between the
syntaxin 1 retained in the ER and the redistributed TI-VAMP and cellubrevin by
co-immunoprecipitation. (4) The effect of the expression of syntaxin 1 on the
redistribution of CD63 suggests that the vesicles containing TI-VAMP and CD63
docked and fused with the syntaxin-1-containing ER membrane. At steady state,
the effect of the expression of syntaxin 1 in the ER on CD63 localization was
only partial (Fig. 8A). This
could be due to the fact that TI-VAMP and CD63 may not necessarily be
transported in the same vesicles all along their trafficking pathway. In
contrast, the inhibition of the expression of CD63 at the plasma membrane was
very strong (Fig. 8B), thus
demonstrating that the trafficking of CD63 was strongly impaired. The
expression of syntaxin 1 in non-neuronal cells was also shown to induce a
relocalization of Golgi markers (Rowe et
al., 2001). However, the redistribution of TI-VAMP and cellubrevin
observed in syntaxin-1-expressing cells (this study) was not merely the result
of the disassembly of the Golgi because Brefeldin A, a drug that induces the
collapse of the Golgi complex (Fujiwara et
al., 1988
), has no effect on the localization of TI-VAMP
(Advani et al., 1999
).
It is noteworthy that, despite the mislocalization of cellubrevin and
TI-VAMP, cells ectopically expressing syntaxin 1 are still capable of
internalizing and recycling transferrin. This indicates that the
mislocalization of syntaxin 1 to the ER does not have pleiotropic effects on
all membrane fusion steps. This result is rather unexpected because it
suggests that a v-SNARE other than cellubrevin participates in transferrin
recycling. In this regard, it is important to note that endobrevin colocalizes
with internalized transferrin (Wong et
al., 1998) (S.M.-A. and T.G., unpublished) as does cellubrevin
(Galli et al., 1994
), which
suggests that cellubrevin and endobrevin may have overlapping functions. The
participation of endobrevin in this transport pathway could explain why the
treatment of cells with tetanus toxin only inhibited one third of the total
release of apo-transferrin (Galli et al.,
1994
) and the lack of a major phenotype in cellubrevin-knockout
mice (Yang et al., 2001
).
Irrespective of the molecular mechanism underlying transferrin recycling in
syntaxin-1-transfected cells, these results support the specificity of the
v-/t-SNARE interaction in vivo and rule out the possibility that the observed
effect on TI-VAMP and cellubrevin could be because of a general defect in
intracellular trafficking.
nSec1 belongs to the Sec/Munc18 (SM) family of proteins
(Rizo and Sudhof, 2002) and is
essential for synaptic vesicle exocytosis
(Verhage et al., 2000
). It
binds tightly to the closed conformation of syntaxin 1
(Dulubova et al., 1999
) and
competes with SNARE complex formation
(Yang et al., 2000
). As
discussed by Rowe et al., nSec1 may act as a chaperone-like protein, allowing
syntaxin 1 to proceed through the secretory pathway by keeping it in a
`closed' conformation, unable to interact with its partner SNAREs
(Rowe et al., 2001
), and
preventing the formation of non-productive SNARE complexes. Our data suggest
that when syntaxin 1 is overexpressed in the absence of nSec1, it may display
an `open' conformation (Dulubova et al.,
1999
) that is unable to exit the ER but able to interact with its
cognate partners, resulting in the final redistribution of its cognate
v-SNAREs to the ER. These results point to the importance of the balance
between syntaxin 1 and nSec1 for the correct functionality of both proteins,
as has been shown to be the case in other systems such as chromaffin cells and
Drosophila (Voets et al.,
2001
; Wu et al.,
1998
). The effect of syntaxin 1 expression reported here may also
explain the blockade of membrane transport observed in these conditions
(Rowe et al., 1999
), because
functional exocytic v-SNAREs would be sequestered in the ER. Moreover, several
studies have revealed the presence of SNARE proteins in lipid rafts and the
importance of maintaining these structures for normal SNARE function
(Chamberlain et al., 2001
;
Lafont et al., 1999
;
Lang et al., 2001
). In MDCK
cells co-transfected with syntaxin 1 and nSec1 there is a fraction of syntaxin
1 associated with lipid rafts that disappears when syntaxin 1 is expressed
alone, resulting in a shift in syntaxin 1 distribution from the plasma
membrane to intracellular structures (Rowe
et al., 2001
). Thus, one possibility that may explain our results
is that the `open' conformation displayed by syntaxin 1 in the absence of
nSec1 is not able to enter special lipid microdomains in the ER. In these
conditions syntaxin 1 would be highly active in SNARE complex formation and
would trap its cognate v-SNAREs in the ER. Regarding this point, there is most
probably a competition for v-SNARE between the `open' syntaxin 1 and
endogenous plasma membrane syntaxins (2, 3, 4). The overexpression of syntaxin
1 together with the fact that endogenous syntaxins are probably in `closed'
conformations because of their interaction with the corresponding SM proteins
(Dulubova et al., 2003
),
displaces the equilibrium towards an interaction with syntaxin 1. Moreover,
the lack of mislocalization of syntaxin 7 upon overexpression in MDCK cells
(Supplementary Fig. 1 available
at
jcs.biologists.org/supplemental),
probably resulting from the expression in these cells of the SM protein
regulating syntaxin 7, strengthens the link between SM-protein-regulated SNARE
complex formation and v-SNARE distribution.
Recent data have shown that, in an in vitro liposome fusion assay, it is
possible to favor the fusion of liposomes containing the yeast v-SNARE Bet1p
over those containing Sft1p by increasing the proportion of a t-SNARE complex
containing Sed5p, Bos1p and Sec22p versus one containing Sed5p, Gos1p and
Ykt6p (Parlati et al., 2002).
An in vivo extension of this observation would imply that increasing the local
concentration of a t-SNARE should increase fusion of cognate v-SNAREs with
that compartment. We found that SNAP-23 partially colocalized and formed a
complex with syntaxin 1 in cells ectopically expressing syntaxin 1 in the ER.
Interestingly, more SNAP-23 was co-immunoprecipitated from extracts of cells
expressing only syntaxin 1 than from those expressing syntaxin 1 and nSec1.
Therefore, in these conditions, the ER membrane fulfilled the essential
conditions to become a target membrane for TI-VAMP and cellubrevin vesicles,
namely the presence of a topologically adequate t-SNARE complex
(Parlati et al., 2000
)
composed of syntaxin 1 and SNAP-23. Our observation that the trafficking of
CD63, a cargo of TI-VAMP, was strongly affected and that this protein
relocalized partially to the syntaxin-1-positive compartment suggests that the
presence of the syntaxin 1/SNAP-23 complex on the ER membrane triggered
docking and fusion of TI-VAMP-containing vesicles.
Altogether, our data strongly suggest that the intracellular distribution
and destination of v-SNAREs are governed, at least in part, by their specific
interaction with cognate t-SNAREs. Interestingly, previous work on Snc1 agrees
with this hypothesis. The yeast exocytic v-SNARE Snc1p, in common with
mammalian exocytic v-SNAREs, is continuously cycling and participates in SNARE
complexes with plasma membrane and endosomal t-SNAREs
(Holthuis et al., 1998). In
mutant cells lacking Tlg1p or Tlg2p, its endosomal partner t-SNAREs, the
steady-state distribution of Snc1p was affected
(Lewis et al., 2000
).
Moreover, Snc1p is also redirected to a haze of transport vesicles in a mutant
yeast strain in which Tlg1p and Tlg2p accumulated on the same structures
(Siniossoglou and Pelham,
2001
). Exocytic v-SNAREs, such as Snc1, synaptobrevin 2,
cellubrevin and TI-VAMP, are continuously cycling between the plasma membrane
and endosomes. Our results suggest that when a t-SNARE is constitutively
active (i.e. syntaxin 1 in the absence of nSec1 in this study) then more
v-/t-SNARE complexes form and the v-SNAREs localize, to a great extent, to the
membrane where the cognate t-SNARE is expressed. In contrast, in normal
conditions (i.e. co-expression of syntaxin 1 and nSec1 in this study or
wild-type fibroblasts) the level of SNARE complexes formed (and therefore
recovered in detergent extracts) is low, and TI-VAMP and cellubrevin localize
to endosomal vesicles. This suggests that the steady-state subcellular
localization of v-SNAREs is the result of equilibrium between two states: one
corresponding to v-SNAREs on the donor vesicles and the other to v-SNAREs in
the target membrane. The lack of nSec1 displaced this equilibrium towards the
second state.
Our findings have important implications for how compartmental specificity is achieved during membrane fusion. Indeed, we have shown that the ectopic expression of syntaxin 1 induces an illegitimate rerouteing of vesicles, the fusion of which is mediated by the cognate v-SNAREs of syntaxin 1, TI-VAMP and cellubrevin. These results show that the highly controlled and specific v-/t-SNARE interaction is essential to define the destination of membrane carriers in vivo in mammalian cells. In conclusion, our results suggest that the exquisite regulation of the v-/t-SNARE interaction that ensures compartmental specificity of membrane fusion is also one of the factors accounting for the accuracy of the dynamic intracellular distribution and destination of v-SNAREs.
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Acknowledgments |
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Footnotes |
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References |
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