1 Programme in Cell Biology The Hospital for Sick Children, University of
Toronto, 555 University Avenue, Toronto, Ontario M5G 1x8 Canada
2 Programme in Developmental Biology, The Hospital for Sick Children, University
of Toronto, 555 University Avenue, Toronto, Ontario M5G 1x8 Canada
3 Department of Biochemistry, University of Toronto, 555 University Avenue,
Toronto, Ontario M5G 1x8 Canada
4 Department of Molecular and Medical Genetics, University of Toronto, 555
University Avenue, Toronto, Ontario M5G 1x8 Canada
* Author for correspondence (e-mail: wtrimble{at}sickkids.on.ca)
Accepted 3 September 2002
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Summary |
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Key words: Golgi, SNARE, Syntaxin, Membrane fusion, Drosophila
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Introduction |
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Originally identified as membrane-associated proteins essential for the
presynaptic release of neurotransmitters, SNAREs comprise syntaxin, SNAP-25
and VAMP/synaptobrevin, each belonging to a protein family with increasing
family members (reviewed by Chen and
Scheller, 2001; Jahn and
Sudhof, 1999
; Rothman,
1994
). Vesicle SNAREs can interact with cognate SNAREs on the
target membrane. In the case of synaptic fusion, syntaxin 1 (on plasma
membranes) and VAMP/synaptobrevin 2 (on synaptic vesicles) each contributes
one helix, whereas SNAP-25 (on plasma membranes) contributes two helices to
form a four-helix bundle. Lying in the core of the bundle are conserved layers
of interacting amino-acid side chains, with the central layer being the most
highly conserved (Sutton et al.,
1998
). Thus, on the basis of whether they provide a glutamine or
arginine at the central layer, SNAREs can be classified as either Q- or
R-SNAREs. Additional structural analysis suggests that a functional SNARE
complex is most probably formed by coiled-coil interactions among three
Q-SNAREs and one R-SNARE that are distributed on apposing membranes
(Antonin et al., 2002
;
Fasshauer et al., 1998
). Such
complexes may be stable enough to survive mild SDS treatment and their
disassembly requires the collaborative actions of the ATPase NSF
(N-ethyl-maleimide-sensitive factor) and its
ligand SNAP (soluble NSF associated
protein) (Otto et al.,
1997
). These observations, together with the fact that the
coiled-coil terminates at the C-terminal transmembrane domain of the SNAREs,
have led to the hypothesis that the formation of the SNARE complex releases
sufficient energy to bring the opposing membranes into close apposition and
thereby promote fusion (Hughson,
1999
). Strong support for this hypothesis comes from the
observation that cognate Q- and R-SNAREs reconstituted separately on
artificial liposomes were sufficient to mediate membrane fusion
(Fukuda et al., 2000
;
Weber et al., 1998
). However,
recent evidence from a number of different systems has suggested that SNARE
complex formation may not constitute the final step of membrane fusion
(reviewed by Mayer, 1999
;
Mayer, 2001
). At least in the
case of yeast vacuolar fusion, additional proteins have been shown to act
after SNARE complex formation (Peters et
al., 1999
; Peters and Mayer,
1998
).
In spite of the debate on the exact role of SNAREs in the final stages of
fusion, it has been generally accepted that the interaction of cognate SNAREs
at least contributes to the specificity of membrane fusion and that most
trafficking events require a different SNARE complex
(Pelham, 2001;
Rothman and Warren, 1994
).
This may explain why there are so many members of the SNARE super-family, with
rather unique but sometimes overlapping distribution patterns along the
secretory and endocytic pathways. Unfortunately, studies on many individual
mammalian SNAREs have yet to provide conclusive evidence for the functional
pairing of cognate SNAREs. This is largely due to two reasons: the
non-specific pairing of SNAREs under in vitro conditions
(Tsui and Banfield, 2000
;
Yang et al., 1999
) and the
difficulty in generating mutant alleles in live animals or cultured tissues to
address the issue in vivo. Drosophila melanogaster, by contrast,
allows great flexibility in genetic manipulation while offering a similar
level of complexity to mammals.
In an attempt to initiate studies on SNARE-mediated membrane trafficking in Drosophila, we screened a fly cDNA library for potential SNAREs using the yeast two-hybrid system. Using this approach, a novel syntaxin isoform that shows significant amino-acid sequence similarity to mammalian syntaxin 16 was identified. dSyx16 is ubiquitously expressed in Drosophila and appears to localize to the Golgi apparatus. Overexpression studies indicate that dSyx16 may selectively regulate Golgi dynamics in the fruitfly.
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Materials and Methods |
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To screen for potential binding partners of dSNAP, a Drosophila melanogaster ovary cDNA library (gift of J. Verdi, University of Western Ontario) constructed downstream of the GAL4 activating motif in the pACT2 vector was used to transform the Y190 strain that expressed the bait. Transformants were plated on His- agar plates from which potential positive clones were selected by monitoring the expression of the His3 reporter gene. For further confirmation, transformants with a His+ phenotype were tested for expression of a second reporter gene, lacZ, using a filter assay for ß-galacosidase activity as recommended (Clontech). To eliminate false positives, candidate transformants were grown in medium with no selection for the bait vector. The transformants that lost the bait were tested again for the lack of lacZ expression.
Plasmid DNA was isolated from yeast, transformed into E. coli and then purified from E. coli for DNA sequencing.
Molecular biology
Drosophila SNAP was cloned by RT-PCR. Total RNA from Oregon
R was isolated using Trizol reagent (Gibco). The first round of cDNA
synthesis was achieved with AMV reverse transcriptase (Promega) using
oligo(dT) as a primer. A subsequent PCR reaction was carried out using
primers: 5'-CATATGGGTGACAACGAACAGAAGGC and
5'-GTCGACTCGCAGATCGGGATCCTCG. The PCR product was subcloned into the
pBluescript SK+ vector (Stratagen) for sequencing.
The partial cDNA of dSyx16, cloned via the yeast two-hybrid
screen, was amplified by PCR using primers:
5'-GAATTCATATGTCTAAGATCAAGCCTAAGCTGG and
5'-CTCGAGCTACTTGCGGTTCTTGCGCTG, or
5'-AGATCTCGAGCTAGAGCTTGGTCAGGATG to generate dSyx1670
to 329 or dSyx1670 to 352
respectively. Each PCR product was subcloned into pBluescript SK+
vector for sequencing. dSyx1670 to 329 was then
subcloned into pGEX-KG vector (Pharmacia) to generate GST-dSyx1670 to
329 for binding assays or into pQE-30 vector (Qiagen) to generate
His-dSyx1670 to 329 for antibody production or into pUAST
(Brand and Perrimon, 1993) for
microinjection. dSyx1670 to 352 was subcloned
into pUAST and pRmHa-3-myc (see below) for microinjection and transient
transfection of cultured cells.
pRmHa-3-myc was constructed by replacing the polycloning site of pRmHa-3 (gift of D. Williams) with the polycloning site of pcDNA3.1-myc, which has a myc epitope at the 5' end. Full-length dSyx16 or the cytoplasmic domain of dSyx16 (dSyx1670 to 329) was coupled downstream from the myc-tag in this vector.
Chromosomal mapping of dSyx16 was conducted by blotting the P1 Drosophila high-density filter (Genome system) with [32P]-labelled dSyx16 cDNA. The position of the positive signal was matched with the chromosomal location, according to instructions provided by the manufacturer. The location of dSyx16 was subsequently confirmed by data from the Berkeley Drosophila Genome Project (http://flybase.bio.indiana.edu/.bin/fbgrmap?fbgene19&id=FBgn0031106).
Binding assays and western blot analysis
BSJ72 expressing GST-dSyx1670 to 329 fusion protein
were lysed using a French press (Sim-Aminco). Fusion protein in the inclusion
body was dissolved with 1% N-lauryl sarcosine in PBS and subsequently treated
with 2% Triton X-100 for 1 hour at 4°C before it was coupled onto
glutathione agarose beads (Sigma). Approximately 200 ng of immobilized
GST-dSyx1670 to 329 or GST (negative control) were then
incubated with specific amounts of recombinant dSNAP
(Mohtashami et al., 2001) in
binding buffer (1xPBS, 0.05% Tween 20, 5 mM EDTA, 100 mM NaCl, and 0.1%
BSA) for 1 hour at 4°C. Following extensive washes with 50 mM HEPES
(pH7.5), 5 mM EDTA, 150 mM NaCl and 0.5% Triton X-100, proteins on the agarose
beads were extracted with 2xSDS sample buffer and subjected to SDS-PAGE.
Western blot analysis was performed with Anti-GST (1:1000) (K. Ross and
W.S.T., unpublished) and anti-dSNAP [1:2000
(Mohtashami et al.,
2001
)].
Oregon R embryos at different developmental stages, third instar larvae, pupae, adults, adult heads, bodies, salivary glands and other imaginal discs dissected from third instar larvae were lysed in homogenization buffer (100 mM Tris, pH 6.8, 20% glycerol, 2% SDS and 5 mM EDTA). Following protein quantification with BCA reagents (Pierce), equal amounts of protein were subjected to 10% SDS-PAGE and western blot analysis using affinity-purified anti-dSyx16 antibody (1:800) that was raised against His-dSyx1670 to 329.
In fractionation studies, Oregon R adults were homogenized in 50 mM HEPES (pH 7.5), 25% sucrose, 200 µM PMSF and 5 mM EDTA. Following centrifugation for 10 minutes at 1200 g, the supernatant was centrifuged at 100,000 g for 1 hour to separate the soluble fraction and the membrane fraction. The membrane pellet was re-suspended and incubated with either H2O, 2 M KCl, 0.2 M Na2CO3 (pH 11-12), 4M urea, 2% Triton X-100 or 2% SDS for 1 hour at 4°C. After centrifugation, both the soluble and insoluble fractions were subjected to SDS-PAGE and western blot analysis.
Immunocytochemistry and transient expression in cultured cells
Schneider cells (S2 cells) were grown on coverslips in Schneider's
Drosophila medium (Gibco) supplemented with 10% FBS overnight before
they were treated with 1.5% DMSO alone (negative control) or 30 µg/ml of
brefeldin A (Sigma) and 1.5% DMSO for 2 hours. Cells were then fixed with 4%
paraformaldehyde in 100 mM Na3PO4 (pH 7.0) for 25
minutes. After incubation with quench buffer (25 mM NH4Cl, 25 mM
glycine, 1xPBS) for 15 minutes, cells were then blocked overnight at
4°C with 2% BSA, 2% normal goat serum in PBT (PBS with 0.1% Triton X-100).
Cells were then incubated for 2 hours with rabbit anti-dSyx16 and mouse
monoclonal anti-p120 (1:500; Calbiochem). Following washes with PBT, cells
were incubated with Alexa-488-conjugated goat anti-rabbit (1:1000; Molecular
Probes) and Cy3-conjugated donkey anti-mouse (1:1000; Molecular Probes) for 1
hour, washed with PBT and then mounted and cleared with DAKO fluorescent
mounting medium.
For transient expression, S2 cells on the coverslip were transiently
transfected overnight using the Ca3(PO4)2
method with various pRmHa-3-myc-dSyx16 constructs, washed with PBS
and re-incubated overnight in 1 mM CuSO4 in Schneider's
Drosophila medium supplemented with 10% FBS medium. The cells were
then fixed and co-stained with Rabbit anti-myc (1:100; Molecular Probes) and
mouse monoclonal anti-P120, which recognizes a 120 kDa Golgi protein
(Stanley et al., 1997).
To examine Golgi morphology, salivary glands of Oregon R were
dissected from third instar larva, fixed in 4% paraformaldehyde and stained
with anti-dSyx16 (1:400). Testes were prepared and immunostained as described
previously (Hime et al.,
1996). To visualize DNA, propidium iodide (5 µg/ml) was used
during the secondary incubation. Images of salivary glands, S2 cells and
testes were captured by a Zeiss LSM510 confocal microscope.
Fly stocks and genetic studies
Stocks were maintained at room temperature on standard cornmeal agar medium
unless otherwise indicated. Visible markers and balancer chromosomes have been
previously described (Lindsley, 1992). Transgenic flies USA-dSyx1670
to 329 and UAS-dSyx1670 to 352 were made by
standard P-element-mediated transformation
(Rubin and Spradling, 1982).
Individual transgenic lines were crossed with C96-GAL4, UAS-dNSF2/TM3,
Ser at room temperature. Adult wings were dissected and placed on glass
slides in a drop of isopropanol and then mounted in a mixture of Canada balsam
and methylsalicylate (Sigma). Images were obtained with a Nikon Optiphot 2
microscope and CCD camera.
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Results and Discussion |
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|
Like human syntaxin 16, Drosophila syntaxin 16 carries at the
C-terminus a 21 amino-acid-long hydrophobic motif, which probably serves as a
transmembrane domain that anchors the protein to the membrane. Adjacent to the
C-terminal hydrophobic motif is a predicted helical domain of about 60 amino
acids with the potential to form a coiled-coil structure
(Fig. 1). This motif is
conserved within the syntaxin family
(Weimbs et al., 1997) and
apparently mediates the interaction of syntaxin with many of its binding
partners (i.e., SNAP, VAMP, SNAP25 etc.). In fact, Drosophila and
human Syx16 share more than 60% amino-acid identity along this domain,
although the overall identity is approximately 35%.
dSyx16 interacts with SNAP and NSF
Two independent approaches were undertaken to test whether dSyx16 indeed
functions as a SNARE. A biochemical approach was first used to examine whether
dSyx16 and dSNAP interact in vitro. Recombinant GST-dSyx16 fusion protein was
attached to glutathione beads and then incubated with purified dSNAP.
Following extensive washes, proteins on the beads were eluted and subjected to
western blot analysis. As shown in Fig.
2, GST-dSyx16 binds to dSNAP in a concentration-dependent fashion,
although an equivalent amount of GST retains no dSNAP. Hence, recombinant
dSNAP binds directly to dSyx16.
|
A second approach to examine the role of dSyx16 involved a genetic approach
in vivo. Each unique SNARE complex would at one stage be disassembled by the
actions of SNAP and NSF so that freed SNAREs can participate in subsequent
rounds of fusion events. Blocking NSF function would block the disassembly of
the SNARE complexes and thereby interfere with membrane trafficking. For
example, overexpression of a dominant-negative form of NSF at the wing margin
resulted in a notch-wing phenotype in adult flies, presumably by inhibiting
secretion/signalling during wing development
[(Stewart et al., 2001);
Fig. 3B].
|
To genetically address whether dSyx16 interacts with NSF, we took advantage
of the observations that overexpression of a syntaxin (with or without its
transmembrane motif) can specifically interfere with the membrane trafficking
step this molecule is responsible for
(Dascher and Balch, 1996;
Hatsuzawa et al., 2000
;
Low et al., 1998
;
Mallard et al., 2002
;
Nagamatsu et al., 1996
;
Nakamura et al., 2000
;
Wu et al., 1998
). Two dSyx16
transgenic flies bearing either amino acids 70 to 329 (UAS-dSyx1670
to 329) or 70 to 352 (UAS-dSyx1670 to 352)
under the control of the GAL4 upstream activating sequence were created.
Overexpression of the dSyx16 protein fragments in the wing margin was
accomplished by crossing the UAS lines with C96-GAL4, which expresses GAL4
protein in the wing margin during wing development. As previously reported
(Stewart et al., 2001
),
ectopic expression of the dominant-negative form of NSF2 under the control of
C96-GAL4 gave rise to mild notches on the wing margin (compare
Fig. 3B with 3A), which were
enhanced by specific alleles of dsyntaxin 1
(Stewart et al., 2001
).
Overexpression of the soluble dSyx16 (amino acid 70 to 329) did not appear to
modify the notch-wing phenotype caused by dominant-negative NSF2 (compare
Fig. 3C and 3B). However,
overexpression of dSyx1670 to 352 significantly enhanced
the notch-wing phenotype (compare Fig. 3D
and 3B). Since the two isoforms were expressed at a similar level
(both isoforms can be distinguished from the wild-type on western blot; data
not shown), it is unlikely that the expression levels are responsible for the
differential effect on wing margin development. One conceivable explanation is
that unlike dSyx1670 to 329, which is dispersed in the
cytosol, dSyx1670 to 352 is delivered to its designated
location where its overexpression may sequester other molecules needed for
fusion. Interestingly, overexpression of dSyx1670 to 352
by itself does not lead to any noticeable defects in the wing margin or
elsewhere (data not shown), indicating that the dominant-negative effect
derived from GAL4-driven overexpression of dSyx16 is not as prominent as that
of NSF2. Nevertheless, both biochemical and genetic studies argue that the
dSyx16 is a functional component of the SNARE complex.
Temporal and spatial localization of dSyx16
To determine the temporal distribution of dSyx16, embryos from Oregon
R were collected every three hours after embryo deposition (AED) and
allowed to develop for up to 24 hours. Embryos were then lysed and equal
amounts of total protein were separated by SDS-PAGE and then immunoblotted
with affinity-purified anti-dSyx16, which was raised in rabbits against
His-dSyx1670 to 329. This antibody recognized a 44 kDa
band from fly lysates, slightly above the predicted molecular mass of the
polypeptide (40 kDa). The band disappeared if the antibody was pre-incubated
with recombinant dSyx16 (data not shown), indicating that the antibody is
specific. Using this antibody, we were able to detect a similar level of
dSyx16 in all embryonic collections (Fig.
4A), suggesting a role for dSyx16 during embryogenesis. To examine
the distribution of dSyx16 in late developmental stages, third instar larvae,
pupae, adults and imaginal discs were collected and subjected to western blot
analysis. As shown in Fig. 4B,
dSyx16 was expressed at all stages examined and appeared to be more abundant
in the adult head than the adult body. In addition, dSyx16 was abundantly
expressed in imaginal discs and other tissues including CNS and salivary
gland, where active membrane trafficking is required during development. That
fact that dSyx16 is ubiquitously expressed throughout the life cycle of
Drosophila is consistent with its potential role as a Golgi SNARE,
which has been suggested by studies on its mammalian homologue Syx16
(Simonsen et al., 1998;
Tang et al., 1998
).
|
Amino-acid sequence analysis (Fig.
1) indicates that dSyx16 may associate with membranes through its
C-terminal hydrophobic domain. To confirm this, adult fly lysates were
separated into soluble and membrane fractions by centrifugation. Subsequent
SDS-PAGE and western blot analysis showed that although dSyx16 was predominant
in the crude membrane fraction, a small portion was present in the soluble
fraction (Fig. 4C). This is
probably because of the fact that dSyx16 carries only two amino acids
following the potential transmembrane domain. Proteins with similar secondary
structure are likely to be deposited into the cytoplasm upon synthesis, since
their membrane insertion is not coupled to translation but requires
alternative mechanisms (Kim et al.,
1999). To further determine whether dSyx16 is an integral membrane
protein, the membrane fraction was treated with KCl,
Na2CO3 (high pH), urea, Triton X-100 and SDS
respectively. KCl and Na2CO3 did not solublize dSyx16,
suggesting that dSyx16 does not bind loosely to the membrane through ionic or
hydrophobic interaction (Fig.
4C). Urea, which disrupts hydrogen bonds, was able to extract a
small fraction of dSyx16, a phenomenon also observed with human syntaxin 18
(Hatsuzawa et al., 2000
). The
fact that most dSyx16 remained urea-insoluble excludes hydrogen bonding as a
significant force that associates dSyx16 with membrane. Meanwhile, like many
mammalian syntaxins (Wong et al.,
1998
), dSyx16 was soluble in SDS and partially soluble with Triton
X-100. Taken together, our data suggest that dSyx16 is probably an integral
membrane protein and its partial insolubility in Triton X-100 suggests that it
may associate with cytoskeletal elements
(Beites et al., 1999
).
We then went on to determine the subcellular localization of dSyx16 in
salivary gland cells. We chose salivary glands because our developmental
western (Fig. 4B) showed that
dSyx 16 was abundant in salivary gland cells, which are much larger than cells
from other tissues. We observed a punctate intracellular staining pattern in
duct cells (Fig. 5), as well as
punctate staining amongst granules in secretory cells (data not shown). It is
evident that in duct cells the distribution pattern of dSyx16 overlaps with
that of p120, a widely used Drosophila Golgi marker, although from
time to time, very small puncta were found to be positive for anti-dSyx16 but
not anti-p120. It is not known whether these fine punctate structures are
simply staining artefacts or specific to duct cells. Similarly, in cultured
Schneider (S2) cells, the staining pattern of dSyx16 matches that of p120,
although the two do not overlap completely
(Fig. 6E). Because p120
colocalizes with ß-cop (Stanley et
al., 1997), a cis-Golgi protein that shuttles between cis-Golgi
and ER, we speculate that dSyx16 may be localized to a compartment adjacent to
the cis-Golgi.
|
|
Human syntaxin 16 has been reported to localize on either the cis-Golgi
(Simonsen et al., 1998) or the
trans-Golgi network (TGN) (Mallard et al.,
2002
). Very recently, a possible role for hSyx16 in
early/recycling endosomes-to-TGN transport has been reported
(Mallard et al., 2002
). In an
attempt to further clarify the localization of dSyx16, we treated S2 cells
with brefeldin A (BFA), a fungal metabolite that disrupts ER-to-Golgi
trafficking. In mammalian systems, this drug causes Golgi markers to
redistribute to the ER (Sciaky et al.,
1997
) and TGN markers to aggregate around the microtubule
organization center. As shown in Fig.
6D, dSyx16 formed aggregates that associated frequently with ring
structures that only became evident upon BFA treatment (compare
Fig. 6C with D). However, to
our surprise, a similar effect on p120 was also observed (compare
Fig. 6A with B). The two
aggregates have distinct morphologies but maintain partial colocalization in
most cells (Fig. 6F).
Therefore, our data support the notion that dSyx16 is a Golgi SNARE localized
in a cisterna adjacent to cis-Golgi that may be the counterpart of the TGN in
mammalian cells.
dSyx16 distribution during cell division
In mammalian cells, most Golgi proteins are absorbed into the ER during
cell division so that they can be partitioned equally into two daughter cells
along with the ER (Roth, 1999;
Seemann et al., 2002
;
Zaal et al., 1999
). This does
not appear to be the case with yeast Golgi, which exists as discrete units
throughout the cytoplasm (Preuss et al.,
1992
). In Drosophila, Golgi membranes do not undergo
morphological changes during early embryogenesis, although dispersion during
mitotic division has been reported in tissue culture cells
(Stanley et al., 1997
). To
investigate how dSyx16-containing membrane behaves during rapid cell division,
we examined the distribution of dSyx16 in Oregon R testes, which are
enriched with germ line cells at different stages of meiosis. In interphase
cells (Fig. 7), anti-dSyx16
highlighted distinctive puncta, whereas anti-p120 frequently decorated ring
structures. The two different structures match well, further supporting
previous observations that dSyx16 and p120 are localized to two
different but adjacent (or even connected) organelles. During anaphase, dSyx16
distribution became much more dispersed, suggesting that the dSyx16-containing
membrane is either vesiculated or redistributed into the ER. Our results are
consistent with the notion that in Drosophila the mechanism of Golgi
inheritance is cell-type specific. It is still unclear why multiple Golgi
partitioning strategies were developed in fruitfly but not other animals.
|
Overexpression of dSyx16 affects Golgi dynamics
To study the role(s) of dSyx16, we chose to use the overexpression
approach. As mentioned earlier, overexpression of a syntaxin may inhibit the
specific membrane fusion step this syntaxin is assigned to without interfering
with other trafficking events. Studies on yeast
(Banfield et al., 1994),
fruitfly (Wu et al., 1998
) and
cultured mammalian cells (Dascher and
Balch, 1996
; Hatsuzawa et al.,
2000
; Low et al.,
1998
; Mallard et al.,
2002
; Nakamura et al.,
2000
) have demonstrated that the inhibitory effect can be obtained
with either the wild-type or the cytosolic form. However, we did not observe
any significant phenotype when we ectopically expressed dSyx16 in a variety of
Drosophila tissues. This is probably due to the relatively low
overexpression level permitted by the UAS-GAL4 system. Therefore, we went on
to transiently express dSyx16 in cultured S2 cells. By placing dSyx16
under the control of the metallothionein promoter, we expected to see a
significant increase in dSyx16 level upon copper induction. Three different
forms of dSyx16 were used to transfect S2 cells, full-length dSyx161
to 352, dSyx1670 to 329 and dSyx1670
to 352. After transfection, cells were induced overnight with 1 mM
CuSO4 before they were fixed and then stained with anti-myc (to
detect transfected cells), anti-p120 or anti-lava. In cells with relatively
low expression levels, myc-dSyx16 maintained partial colocalization with p120
in the Golgi (data not shown). Overexpression of either dSyx16 or
dSyx1670 to 352 caused the dispersal of p120 in more than
60% of the cells, whereas overexpression of the soluble form had no apparent
effect on the Golgi marker (Fig.
8C,F). This suggests that the first 69 amino-acid residues have
little to do with the negative effect caused by overexpression and that the
transmembrane domain is important for the phenotype. We also noticed that when
dSyx1670 to 352 was overexpressed, it was no longer
localized in large peri-nuclear puncta. Instead, it was dispersed in numerous
fine punctate structures throughout the cytoplasm. Interestingly, although
overexpressing dSyx1670 to 352 might affect the
localization of the Golgi marker p120 or even itself, it did not appear to
affect the distribution of lava-lamp (Fig.
8I), another protein known to be localized to the Golgi
(Sisson et al., 2000
). Two
possible scenarios could account for this observation. First, the Golgi
apparatus may still be intact upon dSyx16 overexpression. Thus, the
overexpression experiments did not simply disrupt the entire secretory pathway
but rather had an inhibitory effect on the dynamics of specific Golgi proteins
such as p120. However, since lava-lamp is a peripheral membrane protein
associated with microtubules, we cannot rule out the possibility that anti-lva
could decorate Golgi remnants even after Golgi membranes had been recycled.
Future studies will be aimed at addressing these issues.
|
In yeast and mammals, syntaxin 5 has been shown to function in ER-to-Golgi trafficking. In mammals, syntaxin 16 and syntaxin 6 are thought to be localized to the late Golgi compartments and have recently been shown to receive retrograde transport from the endosomes. Our overexpression studies provided evidence that Drosophila syntaxin16 is likely to be involved in Golgi dynamics but have not precisely defined the role of this protein, because blocking traffic at either side of the Golgi can potentially disturb the distribution of Golgi proteins. Future work is warranted to address this issue as well as the functional relationship between dSyx16 and its partners.
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Acknowledgments |
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