Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway
* Author for correspondence (e-mail: ksandvig{at}radium.uio.no)
Accepted 13 June 2002
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Summary |
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Key words: Ricin, Golgi apparatus, Calcium, Rab9
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Introduction |
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Several lines of evidence suggest the existence of different transport
routes to the Golgi apparatus, one being the well characterized,
Rab9-dependent route from late endosomes that is utilized by the mannose
6-phosphate receptor (M6PR) (Lombardi et
al., 1993; Riederer et al.,
1994
; Itin et al.,
1997
; Itin et al.,
1999
; Nicoziani et al.,
2000
; Miwako et al.,
2001
). An alternative route seems to be used by both TGN38 and
Shiga toxin B-chain transport to the TGN occurs from early endosomes,
possibly via the endocytic recycling compartment
(Ghosh et al., 1998
;
Mallard et al., 1998
). In the
case of Shiga B, this transport may occur through a clathrin- and
Rab11-dependent mechanism (Mallard et al.,
1998
; Wilcke et al.,
2000
). Ricin also seems to be transported to the Golgi apparatus
using a mechanism that differs from that of the M6PR. We have recently found
that ricin transport to the Golgi apparatus is independent of the small GTPase
Rab9 (Iversen et al., 2001
).
Interestingly, this transport is also independent of functional clathrin and
Rab11, suggesting that it differs from that of Shiga B-chain
(Iversen et al., 2001
).
However, the transport mechanism of ricin to the Golgi apparatus has not yet
been characterized. In the present work we describe the study of whether this
transport is regulated by calcium.
Calcium plays an important role in intracellular transport. It is involved
in both ER to Golgi transport (Schwaninger
et al., 1991), intra Golgi transport
(Porat and Elazar, 2000
) and
fusion between late endosomes and lysosomes
(Colombo et al., 1997
;
Peters and Mayer, 1998
;
Holroyd et al., 1999
). To
investigate the effect of calcium on the transport of ricin and M6PR to the
Golgi apparatus, we treated cells with thapsigargin, which specifically
inhibits the ER Ca2+-ATPase
(Thastrup et al., 1990
). This
inhibition prevents calcium reuptake into the ER, resulting in calcium
depletion of the ER and an increased concentration of calcium in the cytosol.
The effect of the calcium ionophore A23187 was also investigated. Both
thapsigargin and A23187 increased the transport of ricin to the Golgi
apparatus, whereas transport of the M6PR was not affected by such treatment.
Interestingly, the increased transport of ricin seen upon
thapsigargin-treatment was strongly inhibited by wortmannin or LY294002,
suggesting that phosphoinositide 3-kinase (PI3-kinase) might be involved in
this mechanism.
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Materials and Methods |
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Cells
MDCK II cells [from W. Hunziker (Garred
et al., 2001)] were maintained in Dulbecco's modified Eagle's
medium (DMEM) supplemented with 5% fetal calf serum (FCS), 0.25 mg/ml
geneticin, 100 units/ml penicillin, 100 µg/ml streptomycin and 2 mM
L-glutamine. The cells were seeded out into 5 cm Petri dishes at densities of
0.6-1.0x106 cells per dish two days before the sulfation
experiments in the same medium but with 10% FCS. For transfection studies,
MDCK II cells were seeded out into 5 cm Petri dishes at a density of
4.5x105 cells per dish. The next day cells were transfected
using FuGENE(TM)-6 (Boehringer Mannheim) according to the procedure given by
the company. For confocal studies MDCK II cells were seeded on coverslips two
days before the experiments. When polarized MDCK II cells were used, cells
were seeded out on polycarbonate filters (Costar Transwell, Sigma; pore size
0.4 µm, diameter 24.5 mm) at a density of 8x105 per filter
and used for experiments 4 days later.
Measurements of cytosolic calcium concentrations
MDCK II cells grown on coverslips were washed twice with buffer (14 mM
NaCl, 2 mM CaCl2, 20 mM Hepes) and incubated with or without 1
µg/ml thapsigargin or 10 µM A23187 for 30 minutes at 37°C in the
same buffer. The cells were then loaded with fluorescent calcium indicators by
incubation with buffer containing Fluo-3 AM-ester (5 µM) and Fura Red
AM-ester (50 µM) for 1 hour at room temperature in the absence or presence
of thapsigargin or A23187 (a ten times lower concentration of Fluo-3 was used
since the fluorescence of Fluo-3 is around 10 times brighter than Fura Red
when exited at 488 nm). To improve loading, the Fluo-3/Fura Red mixture was
first mixed 1:1 with the polyol surfactant Pluronic F-127 (20% in DMSO) before
addition of buffer. The cells were then washed twice with buffer before
incubation for a further 30 minutes in indicator-free buffer with or without
thapsigargin or A23187 to allow complete de-esterification of the
intracellular AM-esters. Confocal imaging was performed using a Leica TCS NT
confocal microscope equipped with a 63x objective and a Kr/Ar laser. The
488 nm line of the laser was used for excitation, and the emitted fluorescence
was acquired at 530 nm (Fluo-3) and >590 nm (Fura Red) using separate
photomultipliers. Images of 1024x1024 pixels were taken, and the
Fluo-3/Fura Red fluorescence ratio of defined areas of the cytosol was
determined after calculating the mean fluorescence level (0-255) of both
channels (probes) using Adobe Photoshop 4.0 imaging software. The Fluo-3/Fura
Red fluorescence ratios were then converted to calcium concentration values by
in vitro calcium calibration using the Calcium Calibration Buffer Kit #2
(Molecular Probes), which contains buffers with known calcium concentrations.
After de-esterification of the AM-esters by chemical hydrolysis (and pH
adjustment to 7.0), the Fluo-3/Fura Red mixture (1:10, as this was the ratio
used for the indicator loading) was added to small aliquots of the different
calcium buffers in the kit. The solutions were then placed on coverslips, and
the mean fluorescence levels at the two emission wavelength were recorded for
each solution. The Fluo-3/Fura Red fluorescence ratios were determined, and
the calibration points were fitted with Grynkiewicz's equation
(Grynkiewicz et al., 1985). The
Fluo-3/Fura Red fluorescence ratio obtained this way was 0.13 in the control
situation, which corresponds to a calcium concentration of
80 nM. In the
thapsigargin- and A23187-treated cells, the cytosolic calcium levels were
elevated, and the ratios were found to be 0.89 and 1.48, respectively,
corresponding to calcium concentrations of
350 nM and
530 nM,
respectively.
Vectors and constructs
The coding region of the mutant Rab9S21N
(Lombardi et al., 1993) was
cloned into the BamHI/EcoRV sites of the expression vector
pcDNA3. The plasmid construct of the 46 kDa cation-dependent M6PR tagged with
polyhistidine, a c-myc epitope and a tyrosine sulfation site (M6PR46-HMY) was
a gift from Suzanne R. Pfeffer.
Endocytosis of ricin
Endocytosis of ricin was measured using the ORIGEN analyzer (IGEN,
Rockville, MD), which uses electrochemiluminescence detection. Ricin was
labeled with N-hydroxysuccinimide ester-activated tris(bipyridine) chelated
ruthenium(II) TAG (IGEN) according to the procedure given by the company and
simultaneously biotinylated with the reducible ImmunoPure NHS-SS-Biotin
(Pierce, Rockford, IL). MDCK II cells were washed with Hepes medium and
preincubated with thapsigargin for 30 minutes at 37°C before TAG- and
biotin-labeled ricin (30 ng/ml) was added to the medium. The incubation was
continued for either 20 minutes or 2 hours. The cells were subsequently washed
twice (5 minutes) with 0.1 M lactose in Hepes medium at 37°C to remove
surface-bound ricin, then once with cold PBS before they were lysed (lysis
buffer: 0.1 M NaCl, 5 mM MgCl2, 50 mM Hepes and 1% Triton X-100).
The cells were centrifuged for 5 minutes at 16,000 g in an
Eppendorf centrifuge to remove the nuclei, and the amount of ricin in the
cleared lysates was measured using streptavidin beads (Dynal, Oslo, Norway)
and ORIGEN Analyzer (IGEN).
Preparation of ricin sulf-2
The modified ricin A-sulf-2, containing a tyrosine sulfation site and
N-glycosylation sites, was produced, purified and reconstituted with ricin
B-chain to form the holotoxin ricin sulf-2 as described previously
(Rapak et al., 1997).
Sulfation of ricin sulf-2
MDCK II cells were washed twice with DMEM without sulfate and incubated
with 0.2 mCi/ml Na235SO4 for 3 hours at
37°C in the same medium but supplemented with 1 mM CaCl2, 2 mM
L-glutamine and 1xnon-essential amino acids (Life Technologies, Paisley,
UK). Thapsigargin or A23187 was added to the medium during the last 30 minutes
of this incubation, then ricin sulf-2 (200 ng/ml) was added and the
incubation was continued for 2 hours. The cells were washed twice (5 minutes)
with 0.1 M lactose in Hepes medium at 37°C to remove surface-bound ricin
sulf-2, then once with cold PBS before they were lysed [lysis buffer: 0.1 M
NaCl, 10 mM Na2HPO4, 1 mM EDTA, 1% Triton X-100
supplemented with a mixture of protease inhibitors (Roche Molecular
Biochemicals, Mannheim, Germany), pH 7.4]. The cells were centrifuged for 10
minutes at 2040 g in an Eppendorf centrifuge to remove the
nuclei, and the cleared lysate was immunoprecipitated with rabbit anti-ricin
antibodies immobilized on protein A-Sepharose beads (Pharmacia, Piscataway,
NJ) overnight at 4°C. The beads were then washed twice with PBS containing
0.35% Triton X-100 before the adsorbed material was analyzed by SDS-PAGE under
reducing conditions.
Sulfation of the mannose 6-phosphate receptor
Sulfation was performed according to the assay described previously
(Itin et al., 1997). MDCK II
cells transfected with M6PR46-HMY (as described above) were grown in DMEM
without sulfate supplemented with 1.8 mM CaCl2, 2 mM L-glutamine
and 1xnon-essential amino acids, 1xMEM amino acids,
1xvitamin solution, 1 mM sodium pyruvate, 10 mM sodium chlorate and 10%
FCS. Subsequently, the cells were washed twice with DMEM without sulfate
supplemented with 1 mM CaCl2, 2 mM L-glutamine and
1xnon-essential amino acids and preincubated with thapsigargin for 30
minutes at 37°C before 0.6 mCi/ml
Na235SO4 was added to the medium. After 3
hours, the cells were washed twice with cold PBS and lysed in lysis buffer
[0.1 M NaCl, 10 mM Na2HPO4, 1 mM EDTA, 1% Triton X-100
supplemented with a mixture of protease inhibitors (Roche Molecular
Biochemicals), pH 7.4] containing 25 mM imidazole. The cells were then
centrifuged for 10 minutes at 2040 g in an Eppendorf
centrifuge to remove the nuclei. The cleared lysate was immunoprecipitated
with nickel agarose beads (Qiagen, Chatsworth, CA) overnight at 4°C, then
the beads were washed four times with lysis buffer containing 25 mM imidazole.
The adsorbed M6PR46-HMY was eluted with 25 mM EDTA in lysis buffer and 25 mM
imidazole and analyzed by SDS-PAGE under reducing conditions.
Sulfation of STxB-Sulf2
The modified version of Shiga toxin B-chain containing sulfation sites
(STxB-Sulf2) was a kind gift from L. Johannes, The Curie Institute,
Paris. MDCK II cells were washed twice with DMEM without sulfate and incubated
with 0.2 mCi/ml Na235SO4 for 3 hours at
37°C in the same medium but supplemented with 1 mM CaCl2, 2 mM
L-glutamine and 1xnon-essential amino acids (Life Technologies).
Thapsigargin or A23187 was added to the medium during the last 30 minutes of
this incubation, then STxB-Sulf2 (2.8 µg/ml) was added and the
incubation was continued for 2 hours. The cells were washed twice with cold
PBS and then lysed [lysis buffer: 0.1 M NaCl, 10 mM
Na2HPO4, 1 mM EDTA, 1% Triton X-100 supplemented with a
mixture of protease inhibitors (Roche Molecular Biochemicals), pH 7.4]. The
cells were centrifuged for 10 minutes at 2040 g in an
Eppendorf centrifuge to remove the nuclei, and the cleared lysate was
immunoprecipitated with rabbit anti-Shiga toxin antibodies immobilized on
protein A-Sepharose beads (Pharmacia, Piscataway, NJ) overnight at 4°C.
The beads were then washed twice with PBS containing 0.35% Triton X-100 before
the adsorbed material was analyzed by SDS-PAGE under reducing conditions.
SDS-PAGE
SDS-PAGE was performed in 12% gels as previously described
(Laemmli, 1970). The gels were
fixed in 4% acetic acid and 27% methanol for 30 minutes and then incubated for
20 minutes in 1 M sodium salicylate, pH 5.8, in 2% glycerol. Kodak XAR-5 films
were exposed to the dried gels at -80°C for autoradiography. Moreover,
signal intensities of the bands were quantified by exposing the gels to
PhosphoImager screens and using the ImageQuant 5.0 software (Amersham
Biosciences, Sunnyvale, CA).
Confocal microscopy studies
MDCK II cells grown on coverslips were washed twice with Hepes medium and
preincubated with thapsigargin for 30 minutes at 37°C before CY3-labeled
ricin B-chain (1 µg/ml) was added. After 2 hours of incubation, the cells
were fixed with 3% paraformaldehyde in PBS, permeabilized with 0.1% Triton
X-100 and blocked with 5% FCS. The Golgi apparatus was then labeled with
rabbit anti-mannosidase II antibodies (from Kelley Moremen, University of
Georgia, Athens, GA) that were visualized by goat anti-rabbit IgG FITC
(Jackson Immunoresearch Laboratories, West Grove, PA). Colocalization of the
CY3-labeled ricin B-chain and the Golgi apparatus was analyzed by a Leica TCS
NT confocal microscope equipped with a 63x objective and a Kr/Ar
laser.
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Results |
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We next wanted to investigate whether it was the elevated cytosolic calcium level or the depletion of calcium in the ER that was responsible for the thapsigargin-induced stimulation of ricin transport to the Golgi apparatus. For this purpose, we investigated the effect of another calcium mobilizing compound, the calcium ionophore A23187. A23187 increases the permeability to calcium of all cellular membranes, thus cells exposed to A23187 in the presence of a normal extracellular calcium concentration will achieve an elevated calcium concentration in the cytosol, as well as in the lumen of the ER. We checked that the calcium level in the cytosol was elevated in our experiments using ratiometric confocal imaging with fluorescent calcium indicators (see Materials and Methods). Only in the absence of extracellular calcium A23187 is able to deplete the ER of calcium. We therefore incubated MDCK II cells with radioactive sulfate in the presence of 10 µM A23187 using medium containing 1 mM calcium. This concentration of A23187 stimulated the transport of ricin to the Golgi apparatus to the same extent as 1 µg/ml thapsigargin (Fig. 3), indicating that it is the elevated cytosolic calcium level that is responsible for the stimulated transport of ricin.
|
Thapsigargin increases the Golgi transport of both apically and
basolaterally internalized ricin in polarized cells
Some intracellular transport routes are regulated in a different manner in
non-polarized and polarized cells. Also, pathways leading from either the
apical or the basolateral pole of polarized cells are differentially regulated
(Llorente et al., 1996;
Llorente et al., 1998
). We
therefore examined whether thapsigargin also affected the transport of ricin
in polarized cells. Thus, MDCK II cells grown on polycarbonate filters were
preincubated for 30 minutes with thapsigargin (1 µg/ml) before ricin sulf-2
was added either to the apical or to the basolateral pole.
Fig. 4 shows that thapsigargin
increased the transport of ricin to the Golgi apparatus both when ricin was
internalized apically or basolaterally. A23187 exerted a similar effect (data
not shown).
|
Ricin B-chain has a more perinuclear location in cells treated with
thapsigargin than in untreated cells
Consistent with the data obtained by the sulfation experiments, we were
also able to visualize by immunofluorescence the increased transport of ricin
to the Golgi apparatus upon thapsigargin treatment. MDCK II cells were
incubated with CY3-labeled ricin B-chain in the presence of thapsigargin (1
µg/ml). As shown in Fig. 5,
ricin B-chain had a more perinuclear location in the cells that were treated
with thapsigargin compared with the control cells (the colocalization of ricin
with mannosidase II was increased by a factor of three in the
thapsigargin-treated cells; from 4.6% (of total internalized ricin) to 14.6%,
as quantified by analyzing the respective images using the Adobe Photoshop 4.0
software). This result supports the idea that a rise in cytosolic calcium
levels leads to an increased transport of ricin to the Golgi apparatus. That
the increase in colocalization is not larger is probably because of ricin
transport to the ER (as seen by an increased glycosylation).
|
Thapsigargin does not increase the Rab9-dependent transport of the
M6PR to the Golgi apparatus
We recently found that the transport of ricin to the Golgi apparatus occurs
independently of Rab9 in HeLa cells, suggesting that ricin is transported to
the Golgi apparatus through another pathway than the one used by M6PR
(Iversen et al., 2001). We
therefore wanted to investigate whether calcium selectively regulated ricin
transport to the Golgi apparatus or whether thapsigargin also increased the
transport of the M6PR. Thus, MDCK II cells were transfected with the
M6PR46-HMY, a plasmid construct of M6PR tagged with polyhistidine, a c-myc
epitope and a tyrosine sulfation site (Fig.
6). To achieve an accumulation of unsulfated M6PR46-HMY in the
cells, protein sulfation was reversibly inhibited by incubating the cells with
10 µM sodium chlorate as described earlier
(Itin et al., 1997
;
Iversen et al., 2001
). Two
days after transfection, the cells were preincubated with thapsigargin for 30
minutes before radioactive sulfate was added to the medium (now sodium
chlorate-free), and the incubation was continued for 3 hours. As shown in
Fig. 6A, the amount of sulfated
M6PR46-HMY was not increased upon thapsigargin treatment, in contrast to what
was observed for ricin sulf-2 (Fig.
6D), suggesting that the transport of ricin and the M6PR are
differentially regulated and that they use different pathways to the Golgi. To
obtain further support for the view that ricin utilizes a Rab9-independent
pathway in MDCK II cells, as observed earlier in HeLa cells, and that the
thapsigargin-induced transport of ricin also occurs independently of Rab9, we
performed ricin sulfation experiments not only on untransfected MDCK II cells
but also on MDCK II cells transfected with the dominant-negative mutant Rab9
(Rab9S21N). As shown in Fig.
6D, ricin transport was stimulated to about the same extent after
transfection with Rab9S21N. To test that the mutant Rab9 was expressed at
sufficiently high levels to inhibit late endosome to Golgi transport of the
M6PR, we also examined the effect of Rab9S21N-transfection in MDCK II cells
cotransfected with the M6PR46-HMY. As shown in
Fig. 6, the transport of the
M6PR was reduced by 54% in Rab9S21N-transfected cells
(Fig. 6B,C), verifying that the
expression of Rab9S21N was high enough to inhibit the Rab9-dependent transport
route. As discussed in an earlier study
(Iversen et al., 2001
), the
reduction in sulfation will, because of newly synthesized receptors passing
through the Golgi apparatus during the incubation with radioactive sulfate,
give an underestimation of the inhibition of the Rab9-dependent transport.
|
Wortmannin and LY294002 inhibit thapsigargin-induced stimulation of
ricin transport to the Golgi apparatus
We next tried to identify any calcium sensor(s) that might be involved in
regulating ricin transport to the Golgi apparatus. We had earlier found that
calmodulin plays a role in the regulation of ricin transport in polarized MDCK
I cells (Llorente et al.,
1996), thus a possible involvement of this calcium-binding protein
in the thapsigargin-induced stimulation of ricin transport was examined.
However, we were not able to suppress the thapsigargin-induced stimulation of
ricin transport by incubating the cells with the calmodulin antagonist W7
(data not shown), suggesting that Ca2+ does not increase the
transport of ricin to the Golgi apparatus via calmodulin. We further
investigated whether PI 3-kinase was involved in the mechanism that regulates
ricin transport, since the lipid products of this kinase are involved in
vesicle-mediated protein transport (reviewed in
Simonsen et al., 2001
). MDCK
II cells incubated with radioactive sulfate were preincubated with increasing
amounts of the PI 3-kinase inhibitor wortmannin before addition of
thapsigargin and ricin sulf-2. Because of the instability of wortmannin
(Woscholski et al., 1994
), it
was added repeatedly during the incubation time to the final concentrations of
100 nM, 1 µM or 10 µM, respectively. As shown in
Fig. 7A, the
thapsigargin-stimulated ricin transport was reduced by 40% in the presence of
100 nM wortmannin (quantified using the ImageQuant 5.0 software). 1 µM
wortmannin inhibited the transport even more (60%), whereas an almost complete
block was observed in the presence of 10 µM of the inhibitor. Control
experiments showed that the reduction in sulfation of ricin sulf-2 by
wortmannin was not due to a reduction in protein sulfation in general or a
reduction in the endocytic uptake of ricin (data not shown). The effect of
wortmannin on the A23187-stimulated ricin transport was also examined and
found to be similar to that of thapsigargin-induced transport (data not
shown). Further confirmation of a possible involvement of PI 3-kinase in the
mechanism that regulates ricin transport to the Golgi apparatus was obtained
by demonstrating a strong inhibition by another PI 3-kinase inhibitor,
LY294002, which is structurally different from wortmannin
(Fig. 7B). Also ricin transport
occurring in the absence of increased cytosolic calcium levels was strongly
reduced by wortmannin and LY294002 (data not shown).
|
We also investigated whether protein phosphorylation is involved in the
mechanism that regulates ricin transport. Since both A23187 and thapsigargin
activate p38 MAPK (Chao et al.,
1992), we investigated a possible involvement of this kinase.
However, none of the MAPK inhibitors used (PD169316 and SB203580) were able to
suppress the thapsigargin-stimulated transport of ricin to the Golgi apparatus
(data not shown), suggesting that MAPK does not play a role in this mechanism.
Also inhibitors of PKC, PKA, MEK and Src-kinase have been tested, but none of
these exerted any effect on the thapsigargin-induced stimulation of ricin
transport to the Golgi apparatus.
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Discussion |
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---|
It now appears that there are several pathways between endosomes and the
Golgi apparatus (Goda and Pfeffer,
1988; Ghosh et al.,
1998
; Mallard et al.,
1998
; Mallet and Maxfield,
1999
; Johannes and Goud,
2000
; Iversen et al.,
2001
; Nichols et al.,
2001
), although the best characterized route is the Rab9-dependent
pathway from late endosomes that is utilized by the M6PR
(Lombardi et al., 1993
;
Riederer et al., 1994
;
Itin et al., 1997
;
Itin et al., 1999
;
Nicoziani et al., 2000
;
Miwako et al., 2001
). In
contrast to the results obtained with ricin, sulfation experiments on cells
transfected with the M6PR46-HMY revealed that the transport of the M6PR to the
Golgi apparatus was not increased upon elevated cytosolic calcium levels.
Furthermore, ricin transport to the Golgi apparatus seems to occur, as
previously found in HeLa cells (Iversen et
al., 2001
), independently of Rab9 in MDCK II cells. The
thapsigargin-induced transport of ricin also seemed to be independent of Rab9
as sulfation was not reduced by transfection of cells with mutant Rab9. In the
same experiment the transport of M6PR in Rab9S21N transfected cells was also
analyzed, and the results confirmed that the expression of mutant Rab9 was
high enough to inhibit the Rab9-dependent transport route to the Golgi
apparatus. Thus, calcium does not seem to regulate protein trafficking to the
Golgi apparatus in general, but seems to selectively stimulate the
Rab9-independent endosome to Golgi pathway used by ricin. Interestingly,
thapsigargin also stimulated sulfation of Shiga B, but only by a factor of
two, suggesting that there are differences in transport mechanisms between
these two toxins. Increased knowledge of intracellular routing of protein
toxins might in the future provide us with sufficient insight to prepare drugs
that prevent intoxication and thereby be used as therapeutics.
In polarized cells, transport of proteins internalized from either the
apical or the basolateral pole can be differentially regulated
(Llorente et al., 1996;
Llorente et al., 1998
).
However, as shown here, the transport of ricin to the Golgi apparatus was
found to be stimulated by the elevated cytosolic calcium level both when ricin
was internalized apically or basolaterally. Thus, the calcium-mediated
regulation of ricin transport to the Golgi apparatus occurs independently of
whether ricin passes through early apical or basolateral endosomes.
The PI 3-kinase inhibitors wortmannin and LY294002 significantly reduced
the thapsigargin-induced transport of ricin. A concentration between 1 and 10
µM of wortmannin was required to obtain strong inhibition. However, several
recent reports describe PI 3-kinases that are sensitive to micromolar levels
of wortmannin only (Jones and Howell,
1997; Warashina,
2000
; Hidaka et al.,
2001
). Thus, the transport of ricin to the Golgi apparatus seems
to be dependent on PI 3-kinase, since low concentrations of wortmannin reduced
ricin transport by as much as 40%.
From the present work we conclude that ricin transport to the Golgi apparatus is regulated by calcium, possibly via PI 3-kinase, and that this regulation is selective since it does not affect the Rab9-dependent M6PR transport. Thus, different pathways operating between endosomes and the Golgi apparatus are regulated differentially.
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Acknowledgments |
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