Download Evidence that the transport of ricin to the cytoplasm is independent

Research Article
3503
Evidence that the transport of ricin to the cytoplasm is
independent of both Rab6A and COPI
Alice Chen, Ramzey J. AbuJarour and Rockford K. Draper*
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: [email protected])
Accepted 30 April 2003
Journal of Cell Science 116, 3503-3510 © 2003 The Company of Biologists Ltd
doi:10.1242/jcs.00641
Summary
Cholera toxin, Shiga toxin and ricin are examples of
protein toxins that require retrograde transport from the
Golgi complex into the endoplasmic reticulum (ER) to
express their cytotoxic activities and different toxins
appear to use different pathways of retrograde transport.
Cholera toxin contains the mammalian retrograde
targeting signal KDEL and is believed to exploit the coat
protein I (COPI) and KDEL receptor-dependent pathway
to go from the Golgi complex to the ER. Shiga toxin,
however, has no KDEL sequence to specify its inclusion in
COPI-coated retrograde vesicles and is believed to use a
recently discovered COPI-independent and Rab6Adependent retrograde pathway to enter the ER. Ricin, like
Shiga toxin, does not contain a KDEL sequence and is
Introduction
Cholera toxin, Shiga toxin and ricin are protein toxins that
damage mammalian cells by a mechanism that includes four
essential events: receptor-mediated endocytosis, retrograde
transport into the lumen of the endoplasmic reticulum (ER),
passage through the ER membrane into the cytoplasm, and
catalytic inactivation of a target substrate in the cytoplasm. The
receptor-binding site of a protein toxin is usually within a
subunit or domain that is distinct from the subunit or domain
that bears the catalytic activity of the toxin. Both cholera toxin
and Shiga toxin belong to the AB5 protein toxin family in
which the A chain is the enzymatic subunit and each of the five
identical B chains bind cell surface receptors. The receptor for
cholera toxin is the ganglioside GM1 and the receptor for Shiga
toxin is the globotriaosyl ceramide Gb3 (Lingwood, 1996;
Spangler, 1992). The A chain of cholera toxin ADP-ribosylates
a regulatory G protein and activates adenylate cyclase, which
elevates the intracellular cAMP level (Spangler, 1992),
whereas the A chain of Shiga toxin arrests protein synthesis by
inactivating 28S ribosomal RNA (Endo and Tsurugi, 1987).
Ricin has a subunit organization different from the AB5 toxins
and contains a single A chain and a single B chain. The B chain
binds glycoconjugates that contain galactose residues so that
either cell surface glycoproteins or glycolipids that contain
galactose can serve as ricin receptors (Sandvig and van Deurs,
2000). The A chain of ricin arrests protein synthesis in target
cells by inactivating 28S ribosomal RNA through an enzymatic
mechanism identical to that of Shiga toxin (Endo et al., 1987;
Endo and Tsurugi, 1987).
After binding to cell surface receptors, cholera toxin, Shiga
therefore a candidate to use the COPI-independent and
Rab6A-dependent pathway of retrograde transport to
access the ER. We measured the effect of the GDPrestricted mutant of Rab6A (Rab6A-T27N) on the
cytotoxic activity of ricin and found that expressing
Rab6A-T27N in cells did not inhibit the cytotoxicity of
ricin, suggesting that ricin enters the cytoplasm by a
retrograde pathway that does not involve Rab6A.
Moreover, ricin still intoxicated cells when Rab6A and
COPI were simultaneously inhibited, implying that ricin
requires neither Rab6A nor COPI to intoxicate cells.
Key words: Ricin, Shiga toxin, Rab6, Retrograde transport, COPI
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 (α-, β-, β′-, γ-, δ-, ε- and ζ-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
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Journal of Cell Science 116 (17)
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.
Materials and Methods
Materials
Ricin, bovine serum albumin and rhodamine-labeled secondary
antibodies were from Sigma Chemical Company (St. Louis, MO,
USA). Cholera toxin was from Calbiochem (La Jolla, CA, USA).
Rabbit antiserum to TGN-38 was generously provided by Dr S.
Milgram (The University of North Carolina, Chapel Hill, NC, USA).
Shiga toxin was purchased from Toxin Technology (Sarasota, FL,
USA). cDNA encoding human Rab6A was provided by Dr W. Maltese
(Medical College of Ohio, Toledo, OH, USA). Rab6A DNA was
digested with EcoRI and BamHI and inserted into the vector pEGFPC2 (Clontech Laboratories, Palo Alto, CA, USA) to generate the
Rab6A-GFP construct. A plasmid containing Rab6A-T27N was
kindly provided by Dr B. Goud (Institut Curie, Paris, France). Tran
35S-label was from ICN Radiochemical (Irvine, CA, USA).
Dulbecco’s modified eagle’s medium (DMEM) was from Irvine
Scientific (Santa Ana, CA, USA). Fetal Bovine serum was from
HyClone (Logan, UT, USA). Intracellular cAMP was measured with
a kit purchased from Amersham Pharmacia Biotech (Piscataway, NJ,
USA).
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-T27N
Cells were transfected with expression plasmids encoding either wildtype 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 2×105 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 TPCKtrypsin 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
GDP-restricted Rab6A does not inhibit ricin
formula was used to determine the fraction of HA proteins that were
sensitive to trypsin and therefore on the cell surface: Trypsin-sensitive
fraction (%)=100×[(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.
3505
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).
Results
The effect of Rab6A-T27N on secretion and on the
cytotoxicity of Shiga toxin and cholera toxin
Expression in HeLa cells of the GDP-restricted form of Rab6A
(Rab6A-T27N) delays the secretion of the influenza virus HA
protein (Martinez et al., 1994) and partially inhibits the
cytotoxic activity of Shiga toxin (White et al., 1999). To verify
that expression of Rab6A-T27N had similar consequences in
Vero cells, we measured the effect of expressing Rab6A-T27N
on both HA secretion and on the sensitivity of the cells to
Shiga toxin. Transfected and control cells were infected with
influenza virus, pulsed with radioactive methionine, and
incubated for an hour to allow transport of HA to the cell
surface. Cells expressing Rab6A-T27N transported less HA to
the cell surface in 60 minutes than control cells (Fig. 1). When
the results of three independent experiments were quantitated,
50%±5 of the HA was on the surface of control cells within 60
minutes compared to 26%±6 for cells expressing Rab6AT27N. These results confirm that Rab6A-T27N impairs
secretion of HA in Vero cells similar to that reported for HeLa
cells (Martinez et al., 1994).
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
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 phosphatebuffered saline and incubated with phosphate-buffered saline
containing 1% BSA for 10 minutes. Primary antibodies to TGN-38
Fig. 1. The effect of Rab6A-T27N on the transport of HA to the
plasma membrane in Vero cells. Vero cells were transfected with or
without Rab6A-T27N for 18 hours, infected with influenza virus,
radiolabeled and then chased for 60 minutes as described in
Materials and Methods. After trypsin treatment, the cells were lysed
and HA proteins were immunoprecipitated and resolved in a 12%
SDS-polyacrylamide gel. Transport to the plasma membrane was
determined by trypsin sensitivity as described in Materials and
Methods. HA0 is intact HA whereas HA1 and HA2 are the large and
small trypsin fragments, respectively.
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Journal of Cell Science 116 (17)
Table 1. The effect of Rab6A-T27N on the action of
cholera toxin in Vero cells
cAMP (fmol well–1)
Plasmid
None
Rab6A-T27N
− cholera toxin
+ cholera toxin
483
313
10 278
9 791
Cells were either mock transfected or transfected with the plasmid
encoding Rab6A-T27N for 18 hours. Cells were then incubated with or
without 1 µg ml–1 cholera toxin on ice for one hour to allow toxin binding.
Samples were shifted to 37°C for another hour to allow time for the toxin to
act. Cells were lysed, and intracellular cAMP levels were directly measured
as described in Materials and Methods. Values are the average from two
independent experiments.
Shiga toxin, but does not affect the pathway of retrograde
transport used by cholera toxin.
Fig. 2. The effect of Rab6A-T27N on the sensitivity of cells to Shiga
toxin and ricin. Vero cells were either mock transfected (䊊) or
transfected with a plasmid encoding mutant Rab6A-T27N (䊐) for 18
hours and treated with various concentrations of Shiga toxin (A) or
ricin (B) for 2.5 hours. Tran 35S-label was added for 30 minutes and
the incorporation of radioactivity into acid-insoluble material was
determined as described in Materials and Methods. Each point is the
average of three independent experiments.
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 Rab6AT27N 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.
(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 GDPrestricted 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
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 ε 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 ε-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 Rab6Adependent pathway is impaired. One explanation for this is that
ricin may use either the COPI-dependent or the Rab6Adependent 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.
GDP-restricted Rab6A does not inhibit ricin
3507
Table 2. The effect of Rab6A-T27N on the action of ricin
in ldlF cells at 34°C and 39.5°C
Plasmid
Rab6A
Rab6A-T27N
Rab6A
Rab6A-T27N
°C
IC50
(ng ml–1)
34
34
39.5
39.5
20
14
11
8
Cells were transiently transfected with plasmids containing either wild-type
or mutant Rab6A for 18 hours at 34°C. One-half of the samples were shifted
to 39.5°C and incubated for another 18 hours. The cells were exposed to
different concentrations of ricin for 4.5 hours. Tran 35S label was added for
the last 30 minutes of the incubation and the incorporation of radioactivity
into acid-insoluble material was determined as described in Materials and
Methods. Values are the average of two independent experiments.
Fig. 3. The effect of Rab6A-T27N on the rate at which ricin first
enters the cytoplasm. Mock-transfected control cells (䊊) or cells
transfected with Rab6A-T27N (䊐) for 18 hours were chilled and
incubated with 100 µg/ml of ricin for an hour to bind the toxin to cell
surface receptors. The cells were raised to 37°C by addition of warm
medium and protein synthesis was assessed at the indicated times as
described in Materials and Methods. The solid (control) and broken
(transfected with Rab6A-T27N) lines were fitted to the data points
by the method of least squares.
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
Fig. 4. The distribution of TGN-38 and Rab6A-GFP in ldlF cells.
Cells were transfected with plasmid DNA encoding Rab6A-GFP and
incubated for 48 hours at 34°C. Cells were left at 34°C (A and B) or
shifted to 39.5°C for 6 hours (C and D). The cells were then fixed
and stained with anti-TGN-38 followed by a rhodamine-labeled
secondary antibody. (A) and (C) show rhodamine-labeled anti-TGN38 and B and D are direct visualization of GFP in the fields shown in
A and C, respectively. The arrows in C and D show examples of
structures that label with both TGN-38 and Rab6A-GFP in ldlF cells
at the high temperature. Scale bar in D: 25 µm.
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 γadaptin 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.
Discussion
Three different controls were done to verify the effects of
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Journal of Cell Science 116 (17)
Table 3. The effect of Rab6A-T27N on the action of
cholera toxin in ldlF cells at 34°C and 39.5°C
Clathrin-independent
Endocytosis
Clathrin-dependent
Endocytosis
cAMP (fmol well–1)
Plasmid
°C
− cholera toxin
+ cholera toxin
Rab6A
Rab6A-T27N
Rab6A
Rab6A-T27N
34
34
39.5
39.5
506
750
260
172
11 263
11 966
3 629
3 382
Cells were transiently transfected with plasmids containing either wild-type
Rab6A or Rab6A-T27N for 18 hours at the permissive temperature. One-half
of the samples were shifted to 39.5°C and incubated for another 6 hours
before addition of 1 µg ml–1 cholera toxin for 18 hours. Cells were lysed, and
intracellular cAMP levels were directly measured as described in Materials
and Methods. Values are the average from two independent experiments.
Caveosomes
Early Endosomes
Rab6A'
Recycling
Endosomes
Rab7
Late
Endosomes
Rab6A'
Rab11
Rab9
TGN
expressing Rab6A-T27N on membrane traffic in Vero cells. First,
the secretion of the influenza virus HA protein was monitored in
cells transfected with a plasmid encoding Rab6A-T27N and an
inhibitory effect on HA protein secretion was observed, as noted
by Martinez et al. (Martinez et al., 1994). Second, to confirm that
Rab6A-T27N interfered with the COPI-independent retrograde
transport pathway, the sensitivity of Vero cells to Shiga toxin was
measured after expression of Rab6A-T27N in the cells. The
cytotoxicity of Shiga toxin was partially inhibited, replicating
results observed with Rab6A-T27N in Hela cells (Girod et al.,
1999). Finally, we measured the effect of Rab6A-T27N on
cholera toxin, a toxin that contains a KDEL sequence and which
should not need the Rab6A-dependent pathway to intoxicate
cells. Expressing Rab6A-T27N did not inhibit the action of
cholera toxin in Vero cells. These controls are evidence that
expressing Rab6A-T27N interfered with functions believed to
depend on Rab6A, but not on functions independent of Rab6A.
The effect of Rab6A-T27N on the cytotoxicity of ricin was then
assessed and the major observation here is that neither the IC50
for ricin, nor the rate at which ricin intoxicated cells, was affected
in cells expressing the GDP-restricted derivative of Rab6A.
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 GDPrestricted 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
Golgi
COPI-independent
Rab6A-dependent
COPI-dependent
Rab6A-independent
Endoplasmic Reticulum
Fig. 5. Rab proteins involved in retrograde transport from endosomes
to the ER. Toxins enter cells either by clathrin-independent or
clathrin-dependent endocytosis. Initial pathways of uptake converge
on early endosomes, except for the caveosome pathway which
apparently bypasses the typical endosomal system in transporting
material to the ER. One pathway from early endosomes to the TGN
is via late endosomes that use Rab7 and Rab9 in sequential steps.
Another pathway to the TGN is via recycling endosomes and
involves Rab6A′ and Rab11 in different steps. Rab6A′ may also
participate in the direct transport of material from early endosomes
to the TGN. Two pathways are proposed to transport material from
the TGN-Golgi complex to the ER, the COPI-dependent pathway
that is regulated by Rab6A and the COPI-independent pathway that
is not believed to require Rab6A.
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
GDP-restricted Rab6A does not inhibit ricin
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 COPIdependent 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.
This work partially fulfills the requirement for the Ph.D. degree for
A. Chen. We thank Dr M. Krieger for providing ldlF cells, Dr S.
Milgram for anti-TGN-38, Dr W. Maltese for cDNA encoding Rab6A,
and Dr B. Goud for cDNA encoding Rab6A-T27N. We thank C.
Mikoryak for comments on the manuscript. We are also indebted to
Dr M. Roth for assistance with influenza virus. This work was
supported in part by the National Institutes of Health (GM34297) and
the American Heart Association, Texas Affiliate (0150713Y).
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