The Molecular and Cell Biology Department, FO31, The University of Texas at Dallas, Box 830688, Richardson, TX 75083-0688, USA
* Author for correspondence (e-mail: draper{at}utdallas.edu)
Accepted 30 April 2003
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Summary |
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Key words: Ricin, Shiga toxin, Rab6, Retrograde transport, COPI
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Introduction |
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After binding to cell surface receptors, cholera toxin, Shiga toxin and
ricin are endocytosed into vesicles and use pathways of retrograde transport
to reach the lumen of the ER (Lencer et
al., 1999; Lord and Roberts,
1998
; Majoul et al.,
1996
; Rapak et al.,
1997
; Sandvig et al.,
2002
). Once in the ER, there is evidence that the catalytic chains
of the toxins pass into the cytoplasm by reverse transport through the Sec61p
translocon, the same protein complex used by secretory and membrane proteins
to enter the ER from the cytoplasm
(Koopmann et al., 2000
;
Schmitz et al., 2000
;
Simpson et al., 1999
;
Wesche et al., 1999
).
Protein toxins that exploit pathways of retrograde transport from the
plasma membrane to the ER have been used as model systems to characterize the
transport pathways. The best understood pathway of retrograde transport
involves coat protein I (COPI), a large protein complex that is primarily
found on vesicles budding from Golgi membranes
(Cosson and Letourneur, 1997;
Lippincott-Schwartz et al.,
1998
; Rothman and Wieland,
1996
). The major component of COPI is coatomer, a cytoplasmic
protein complex that contains seven subunits (
-, ß-,
ß'-,
-,
-,
- and
-COP)
(Scales et al., 2000
).
Recruitment of COPI onto membranes depends on the activity of ADP ribosylation
factor 1 (ARF1), a small GTPase protein, and its effectors
(Aoe et al., 1997
;
Aoe et al., 1998
). Together,
coatomer and ARF1 constitute COPI. One function of COPI is to select proteins
in the Golgi apparatus for retrograde transport to the ER. Proteins destined
for Golgi to ER transport carry amino acid signals that allow them to
interact, either directly or indirectly, with COPI and be included in vesicles
that bud at sites coated by COPI. One well-characterized amino acid signal
specifying retrograde transport is the KDEL sequence present on the C-terminus
of soluble proteins that are residents of the ER. These proteins normally
function within the ER, but if they escape to the Golgi, they are returned to
the ER via COPI-coated vesicles. A major insight into the retrograde transport
of protein toxins was that some toxins, such as cholera toxin, contain the
KDEL sequence that allows them to be transported in COPI-coated vesicles from
the Golgi to the ER (Lencer et al.,
1995
; Majoul et al.,
1996
).
Although cholera toxin contains a KDEL sequence, other toxins that require
retrograde transport to reach the ER, such as ricin and Shiga toxin, do not
contain this amino acid motif. In the absence of a known retrograde signal on
these toxins, the mechanism by which they access the ER had been unknown.
Recently, however, a second pathway of retrograde transport that is
independent of COPI (Girod et al.,
1999) was discovered, based in part on the study of Shiga toxin.
This work suggests that toxins bearing KDEL motifs enter the ER by the
classical COPI-dependent pathway and those toxins without KDEL motifs engage
the COPI-independent pathway.
The COPI-independent pathway appears to be regulated by the small GTP
binding protein Rab6A (Girod et al.,
1999; White et al.,
1999
). Rab6A is one of two Rab6 isoforms ubiquitously expressed in
mammalian cells and is associated with medial and
trans-Golgi cisternae as well as the trans Golgi Network
(TGN) (Antony et al., 1992
;
Echard et al., 2000
). Evidence
that Rab6A functions in COPI-independent retrograde transport includes the
observation that expression in cells of a GDP-restricted form of Rab6A
(Rab6A-T27N) inhibits the transport of Shiga toxin to the ER and reduces the
cytotoxic activity of Shiga toxin (Girod
et al., 1999
). It is not clear, however, whether other toxins
lacking a KDEL signal also use the Rab6A-dependent pathway to enter the ER. We
measured the effect of the Rab6A-T27N mutant on the cytotoxic activity of
ricin, which has no KDEL sequence. We also studied the effect of expressing
Rab6A-T27N on the action of ricin under circumstances in which the
COPI-dependent pathway of retrograde transport was inhibited. Our data
suggests that the transport of ricin to the ER is independent of both Rab6A
and COPI.
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Materials and Methods |
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Cell culture
LdlF cells, originally derived from Chinese hamster ovary (CHO) K1 cells
(Guo et al., 1996;
Guo et al., 1994
;
Hobbie et al., 1994
) were
provided by Dr Monty Krieger (Massachusetts Institute of Technology,
Cambridge, MA, USA). Vero cells were obtained from the American Type Culture
Collection (Manassas, VA, USA). All cells were routinely grown in DMEM
supplemented with 10 mM HEPES and 5% fetal bovine serum in incubators with 90%
air and 10% CO2 at either 34°C, 37°C or 39.5°C as
required.
Expression of Rab6A-T27Nm
Cells were transfected with expression plasmids encoding either wild-type
Rab6A or mutant Rab6A (Rab6A-T27N) using LipofectAMINE 2000 (Life
Technologies, Inc., Rockville, MD, USA) following the protocol provided by the
vendor. In brief, cells were plated in 24-well plates at a density of
2x105 cells per well one day before a transfection. To
transfect cells, 1 µg of plasmid DNA was added to 50 µl of the
transfection medium (serum-free DMEM) and 2 µl of LipofectAMINE 2000 was
added to another 50 µl of transfection medium for 5 minutes incubation at
room temperature. The samples containing the plasmid and the LipofectAMINE
2000 were mixed and incubated at room temperature for another 30 minutes.
Cells were washed with phosphate-buffered saline to remove residual serum and
500 µl of serum-free DMEM was added to each well. Mixed transfection medium
(100 µl) containing plasmid and LipofectAMINE 2000 was then added to each
well and incubated for 18 to 24 hours to allow expression of the gene encoded
on the plasmid.
To measure the efficiency of transfection, an additional set of cells were prepared at the same time for immunofluorescence microscopy and stained with a primary antibody to the protein of interest. The percentage of cells in several fields that were positive for the protein of interest was the transfection efficiency and was between 60% and 75% of the total cells.
Analysis of influenza virus HA protein transport
The effect of Rab6A-T27N on the transport of influenza virus hemagglutinin
protein (HA) from the ER to the cell surface was measured by incorporation of
radioactivity from Tran 35S-label into HA as previously described
(Hu et al., 1999). In brief,
cells were transfected with plasmid encoding wild-type Rab6A or the mutant
Rab6A-T27N one day before an experiment. Vero cells were infected with
influenza virus (X31) at 37°C for 30 minutes, washed and incubated for a
further 3.5 hours. LdlF cells were infected with influenza virus (Japan) at
34°C for one hour, washed and incubated for 4 hours. Subsequently, all
cells were incubated for 15 minutes with assay medium containing 100 µCi/ml
of Tran 35S-label. The radioactive medium was then removed and 0.2
ml of fresh assay medium was added for further incubation time as indicated in
the Figures and Tables.
To assess the appearance of HA on the cell surface, cells were treated with extracellular trypsin, which cleaves surface HA into two fragments. Radiolabeled cells were rinsed once with PBS and treated on ice with phosphate-buffered saline containing 100 µg/ml TPCK-trypsin for 30 minutes. Soybean trypsin inhibitor (100 µg/ml) was then added for 10 minutes before the cells were lysed with lysis buffer. HA (X31) was immunoprecipitated overnight with mouse monoclonal antibody FC125 while HA (Japan) immunoprecipitated with polyclonal anti-HA. The immunoprecipitates were incubated at 65°C for 30 minutes in 50 mM sodium citrate, pH 5.5, containing 0.1% SDS, and each sample was mixed with sample buffer before electrophoresis in a 12% SDS-polyacrylamide gel. Radiolabeled protein bands were scanned by PhosphorImaging with the STORM system and quantitated by ImageQuant 5.0 (Molecular Dynamics, Sunnyvale, CA, USA). HA0 is intact HA, HA1 is the large trypsin fragment and HA2 is the small trypsin fragment. The following formula was used to determine the fraction of HA proteins that were sensitive to trypsin and therefore on the cell surface: Trypsin-sensitive fraction (%)=100x[(HA1 + HA2)/(HA0 + HA1 + HA2)]. Three independent experiments were performed with Vero cells and the results are presented as the mean±the standard error of the mean (s.e.m.).
Protein synthesis assay
Protein synthesis was measured by the incorporation of radioactivity from
Tran 35S-label into acid insoluble protein essentially as described
previously (Hu et al., 1999).
Cells were transfected as indicated in Table legends and a toxin was added at
different concentrations for the indicated times. Tran 35S-label (1
µCi/ml) was added for 30 minutes and the cells were washed, lysed, and the
lysate spotted within one-inch squares defined by gridlines drawn on filter
paper. The filter paper was incubated in 5% trichloroacetic acid containing
0.5 mg/ml methionine for 30 minutes at room temperature, washed twice for 5
minutes in 100% ethanol, and dried. Radioactivity within each square of the
grid was measured and quantitated with a PhosphorImager from Molecular
Dynamics. The IC50 value is defined as the concentration of toxin
required to inhibit protein synthesis by 50%. Controls in these experiments
were either cells carried through the transfection procedure without plasmid
(mock-transfected cells) or cells transfected with plasmid encoding wild-type
Rab6A, as indicated in the Figure legends. There was no difference in the
response to toxins of mock-transfected cells and cells expressing wild-type
Rab6A. For experiments done three or more times, the
IC50±s.e.m. is presented.
Kinetics of protein synthesis inhibition by ricin
The rate of ricin intoxication was measured by assessing the incorporation
of radioactivity from Tran 35S-label into acid insoluble protein as
described previously (Bau and Draper,
1993) with modifications. Cells were transfected as indicated in
Fig. 3 for 18 hours before an
experiment. On the day of the experiment, the cells were chilled at 0°C
and incubated with DMEM (lacking methionine) in the presence of ricin (100
µg/ml) for an hour to allow toxin binding to cell surface receptors.
Intoxication was initiated by replacing the cold medium with pre-warmed assay
medium and incubation was continued at 37°C. Tran 35S-label (10
µCi/ml) was added for 10 minutes at desired time points. The cells were
washed, lysed and radioactivity measured as described in the preceding
section.
|
Cholera toxin assays
The effect of cholera toxin on cAMP levels in Vero cells and ldlF cells was
directly measured. Cells in 24-well culture plates were transfected with a
plasmid containing the protein of interest and incubated for 18 hours to
express the protein. Cells were then incubated with or without 1 µg/ml
cholera toxin as indicated in the Table legends. Cells were lysed and
intracellular cAMP levels were directly measured with an immunological assay
according to instructions supplied with the Amersham Pharmacia Biotech assay
kit.
Immunofluorescence microscopy
To transiently express Rab6A-GFP, ldlF cells were transfected with plasmid
using LipofectAMINE PLUS (Life Technologies, Rockville, MD, USA) following the
protocol provided by the vendor. After 48 hours at 34°C, half the samples
were shifted to the restrictive temperature of 39.5°C. For indirect
immunofluorescence, cells were fixed in 4% paraformaldehyde and permeabilized
by 0.2% Saponin for 10 minutes. Samples were then washed 3 times with
phosphate-buffered saline and incubated with phosphate-buffered saline
containing 1% BSA for 10 minutes. Primary antibodies to TGN-38 were incubated
with fixed and permeabilized cells for 30 minutes at room temperature, washed
as in the previous sentence, followed by addition of rhodamine-labeled
secondary antibody and a final washing.
Coverslips were mounted and viewed with a Nikon TE300 microscope equipped
with epi-illuminated fluorescence and a 60X lens (NA=1.4) as
previously described (Chen et al.,
2002). Images were obtained with a MicroMax digital camera
(Princeton Instruments, Trenton, NJ, USA). Pixel intensities were scaled and
images were assembled in Adobe Photoshop 5.5 (Adobe Systems, Inc., San Jose,
CA, USA).
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Results |
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The effect of Rab6A-T27N on the sensitivity of cells to Shiga toxin was
assessed by measuring protein synthesis in transfected and control Vero cells
as a function of toxin concentration. Cells transfected with the plasmid
encoding Rab6A-T27N displayed moderate resistance to Shiga toxin
(Fig. 2A). The average
concentration of Shiga toxin required to reduce protein synthesis by 50%
(IC50) in three independent experiments was 9±2 ng/ml for
control cells and 32±2 ng/ml for cells expressing Rab6A-T27N. Thus,
expressing the GDP-restricted form of Rab6A partially inhibited the
cytotoxicity of Shiga toxin in Vero cells, consistent with previous results in
HeLa cells (White et al.,
1999).
|
Cholera toxin is believed to access the lumen of the ER by a pathway of retrograde transport that is independent of Rab6A; therefore, expressing Rab6A-T27N is not expected to interfere with the action of cholera toxin. To test this, cells making Rab6A-T27N and control cells were incubated with or without 1 µg/ml of cholera toxin and the level of cytoplasmic cAMP was measured. There was no discernible influence of Rab6-T27N on the ability of cholera toxin to elevate cAMP levels (Table 1) in Vero cells. Altogether, the results in this section suggest that Rab6A-T27N impairs secretion, impairs the COPI-independent retrograde transport pathway used by Shiga toxin, but does not affect the pathway of retrograde transport used by cholera toxin.
|
The effect of Rab6A-T27N on the cytotoxicity of ricin
The inhibition of protein synthesis as a function of ricin concentration
for control cells and cells expressing Rab6A-T27N was nearly identical
(Fig. 2B), yielding
IC50 values for ricin of 15±7 ng/ml and 22±4 ng/ml
for control cells and cells expressing Rab6A-T27N, respectively. To further
characterize the effect of Rab6A-T27N on the entry of ricin into Vero cells,
we compared the rate at which ricin is transported from the cell surface to
the cytoplasm in transfected and control cells. Cells were chilled, incubated
with high concentrations of ricin to saturate cell surface receptors, returned
to 37°C, and protein synthesis was assessed at different times. A plot of
the logarithm of the inhibition of protein synthesis versus time yields a
straight line in this type of analysis and extrapolation of the graph to no
inhibition of protein synthesis provides the minimum time required for the
first burst of toxin molecules to reach the cytoplasm
(Neville and Hudson, 1986).
The time required for ricin to reach the cytoplasm of both control cells and
cells expressing Rab6A-T27N was indistinguishable, approximately 30 minutes
(Fig. 3). Thus, expression of
Rab6A-T27N had no significant effect on either the IC50 for ricin
or the time required for ricin to enter the cytoplasm and inhibit protein
synthesis.
The effect of simultaneous inhibition of COPI and Rab6A on the action
of ricin and cholera toxin
We recently noted that ldlF cells, a strain of CHO cells that carries a
temperature-sensitive mutation in the subunit of COPI, were sensitive
to ricin at the restrictive temperature
(Chen et al., 2002
). This
suggested that ricin reaches the cytoplasm by a pathway that does not require
-COP, such as the Rab6A-dependent pathway used by Shiga toxin. However,
the results with Rab6A-T27N in the previous section suggest that ricin still
reaches the cytoplasm when the Rab6A-dependent pathway is impaired. One
explanation for this is that ricin may use either the COPI-dependent or the
Rab6A-dependent pathway, and when one is blocked, the toxin can still access
the cytoplasm by the other pathway. To test this, we measured sensitivity of
cells to ricin under conditions in which both the COPI-dependent and
Rab6A-dependent pathways should be inhibited by expressing Rab6A-T27N in ldlF
cells at the restrictive temperature.
We first characterized the expression of wild-type Rab6A in ldlF cells at
permissive and restrictive temperatures by immunofluorescence microscopy.
Rab6A was imaged in cells transfected with a plasmid expressing Rab6A-GFP
followed by staining for TGN-38 to co-visualize the TGN. In ldlF cells at the
permissive temperature, TGN-38 co-localized closely with Rab6A-GFP in
transfected cells (Fig. 4A,B). At the high temperature, TGN-38 was present in a variety of perinuclear
structures (Fig. 4C),
suggesting that TGN-like membranes persisted in the mutant cells at the high
temperature, consistent with previous results that similar structures
containing -adaptin were present in the ldlF cells at high temperature
(Chen et al., 2002
). The
structures that contained TGN38 in ldlF cells at high temperature also stained
for Rab6A-GFP (Fig. 4D). These
data demonstrate that wild-type Rab6A is expressed in transfected ldlF cells
and that it associates with perinuclear membranes that also bind TGN-38 at
both permissive and restrictive temperatures. We also characterized the effect
of Rab6A-T27N on secretion in ldlF cells to ensure that the
dominant-inhibitory mutant was having the expected effect on secretory
function. LdlF cells were transfected with plasmid encoding Rab6A-T27N,
infected with influenza virus at 34°C, labeled with Tran
35S-label, and the extent of HA on the cell surface was compared to
cells that had not been transfected. Transfected cells had a 46% reduction in
the amount of HA secreted compared to control cells, consistent with the
effects of Rab6A-T27N on retarding secretion in HeLa and Vero cells.
|
The effect of Rab6A-T27N on the sensitivity of ldlF cells to ricin is shown
in Table 2. The IC50
for ricin at the permissive temperature of 34°C in the absence of
Rab6A-T27N was 20 ng/ml and in the presence of Rab6A-T27N was 14 ng/ml. These
control data support the results with Vero cells that expression of Rab6A-T27N
does not inhibit the access of ricin to the cytoplasm. At the restrictive
temperature, the IC50 values for ricin in the absence and presence
of Rab6A-T27N were 11 ng/ml and 8 ng/ml, respectively, suggesting that ricin
efficiently reaches the cytoplasm even when the functions of both COPI and
Rab6A are impaired. Similar experiments were done with cholera toxin in the
ldlF cell system (Table 3).
Expression of Rab6A-T27N at 34°C did not affect the response of ldlF cells
to cholera toxin. The response of the cells to cholera toxin is partially
reduced by incubation at 39.5°C, as previously reported
(Chen et al., 2002), and
expression of Rab6A-T27N did not affect the residual response. Thus, cholera
toxin still reaches the cytosol of ldlF cells regardless of whether Rab6A
function is impaired. The effects of Shiga toxin could not be tested in this
system because neither LdlF cells nor their parental CHO cell line are
sensitive to Shiga toxin.
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Discussion |
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There are two isoforms of Rab6 in mammalian cells generated by alternative
splicing, Rab6A and Rab6A' (also called Rab6C), that differ by only
three amino acids (Echard et al.,
2000). Interestingly, there is evidence that the two isoforms have
different functions. Rab6A is implicated in regulating the COPI-independent
retrograde transport pathway from the Golgi complex to the ER because
Rab6A-T27N inhibits this pathway (Girod et
al., 1999
; White et al.,
1999
). Our observation that Rab6A-T27N failed to interfere with
the action of ricin in either Vero cells or ldlF cells argues that ricin does
not require the COPI-independent and Rab6A-dependent pathway used by Shiga
toxin to go from the Golgi to the ER. Rab6A' is believed to participate
in transport from early/recycling endosomes to the TGN because
Rab6A'-T27N, and antibodies to Rab6A', inhibit this pathway
(Mallard et al., 2002
). It is
difficult, however, to know whether a GDP-restricted mutant interferes with
either Rab6A or Rab6A' because, with the exception of Rabkinesin 6, all
known Rab6 binding proteins interact with both Rab6A and Rab6A'
(Echard et al., 2000
).
Consequently, the GDP-restricted mutant of either one of the two Rab6 isoforms
could sequester essential factors needed by the other isoform and it has been
found that expressing Rab6A-T27N in HeLa cells inhibited the transport of
Shiga toxin from endosomes to the TGN, a pathway believed to require
Rab6A', not Rab6A (Mallard et al.,
2002
). Thus, our data is consistent with the possibility that
ricin does not require the early/recycling endosome-to-TGN pathway controlled
by Rab6A'. This prospect has interesting implications because it has
previously been shown that ricin transport to the Golgi is independent of Rab9
and Rab11 (Iversen et al.,
2001
). Because the only known pathways from endosomes to the TGN
involve either Rab9 or Rab11 or Rab6A', the pathway ricin uses to reach
the TGN is unidentified (see Fig.
5 for an overview of Rab proteins and pathways of retrograde
membrane traffic).
|
What Rab6A-independent pathway of retrograde transport does ricin use to go
from the Golgi complex to the ER? It is conceivable that ricin uses the same
COPI-independent pathway as Shiga toxin, but that Rab6A is not essential for
ricin to move through this pathway. This would imply that some ligands, such
as Shiga toxin, require Rab6A to engage this pathway whereas others, perhaps
ricin, do not. A second possibility is that ricin uses the established
COPI-dependent pathway upon binding to galactose residues of a carrier
glycoprotein that bears the KDEL motif specifying inclusion in COPI-coated
vesicles. However, interfering with COPI function in two different ways fails
to protect cells from ricin (Chen et al.,
2002), suggesting that the COPI-dependent pathway is not essential
for ricin to access the cytoplasm. It is also conceivable that ricin can use
more than one pathway of retrograde transport and when one pathway is
inhibited, another is still available. To test this, we simultaneously
inhibited COPI and Rab6A by raising the temperature in ldlF cells that were
expressing Rab6A-T27N. Ricin still intoxicated the cells when both pathways
were impaired, a significant observation because it suggests that neither the
COPI-dependent nor the COPI-independent pathway is essential for the transport
of ricin to the cytoplasm. Interestingly, cholera toxin still had residual
activity when both pathways were impaired, suggesting the availability of a
pathway independent of COPI and Rab6A for this toxin as well.
It is also possible that ricin reaches the ER by a caveolar pathway. For
example, the transit of SV-40 virus to the ER via a cytoplasmic organelle
termed the 'caveosome' has recently been described
(Pelkmans et al., 2001;
Pelkmans et al., 2002
). In
addition, caveolin-positive endosomes have been implicated in the delivery of
cholera toxin to the Golgi complex from which it may access the ER
(Nichols, 2002
). Ricin
transport is complicated by the likelihood that multiple pathways participate
in the uptake of this toxin and it is difficult to identify pathways that
directly deliver ricin to the ER. To address this, we are continuing to study
conditions that simultaneously inhibit two or more transport pathways in the
event that this will isolate the most important pathways required for ricin to
reach the ER.
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Acknowledgments |
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