pH-independent retrograde targeting of glycolipids to the
Golgi complex
Florencia B.
Schapiro1,2,
Clifford
Lingwood3,4,5,
Wendy
Furuya1, and
Sergio
Grinstein1,2
Divisions of 1 Cell Biology and
3 Microbiology, Research
Institute, Hospital for Sick Children, and Departments of
2 Biochemistry,
4 Microbiology, and
5 Clinical Biochemistry,
University of Toronto, Toronto, Ontario, Canada M5G 1X8
 |
ABSTRACT |
A small fraction
of the molecules internalized by endocytosis reaches the Golgi complex
through a retrograde pathway that is poorly understood. In the present
work, we used bacterial toxins to study the retrograde pathway in Vero
cells. The recombinant B subunit of verotoxin 1B (VT1B)
was labeled with fluorescein to monitor its progress
within the cell by confocal microscopy. This toxin, which binds
specifically to the glycolipid globotriaosyl ceramide, entered
endosomes by both clathrin-dependent and -independent pathways,
reaching the Golgi complex. Once internalized, the toxin-receptor complex did not recycle back to the plasma membrane. The kinetics of
internalization and the subcellular distribution of VT1B were virtually
identical to those of another glycolipid-binding toxin, the B subunit
of cholera toxin (CTB). Retrograde transport of VT1B and CTB was
unaffected by addition of weak bases in combination with concanamycin,
a vacuolar-type ATPase inhibitor. Ratio imaging confirmed that these
agents neutralized the luminal pH of the compartments where the toxin
was located. Therefore, the retrograde transport of glycolipids differs
from that of proteins like furin and TGN38, which require an acidic
luminal pH. Additional experiments indicated that the glycolipid
receptors of VT1B and CTB are internalized independently and not as
part of lipid "rafts" and that internalization is cytochalasin
insensitive. We conclude that glycolipids utilize a unique,
pH-independent retrograde pathway to reach compartments of the
secretory system and that assembly of F-actin is not required for this
process.
cholera toxin; trans-Golgi
network; proton pump; vacuolar-type adenosinetriphosphatase; clathrin
 |
INTRODUCTION |
SURFACE RECEPTORS TOGETHER with their ligands, as well
as other membrane proteins and fluid phase solutes, enter the cells via
clathrin-coated or uncoated vesicles that converge in early endosomes.
Most of the receptors and a fraction of the solutes are rapidly
recycled back to the plasma membrane. In contrast, downregulated
receptors, other membrane proteins, and the remaining solutes are
delivered from the early to late endosomes and subsequently to
lysosomes, where they are degraded. The acidic pH of these compartments, which is maintained by vacuolar-type ATPases (V-ATPases) (12), plays an essential role in the endocytic process. Indeed, dissipation of pH gradients by means of ionophores or weak bases has
been shown to prevent fusion between early and late endosomes (6) as
well as between late endosomes and lysosomes (35).
Although the recycling and lysosomal delivery of internalized molecules
have been extensively investigated, much less is known about the
retrograde transport of surface molecules to the secretory pathway.
Recent studies have revealed the existence of less prominent routes
involved in the delivery of (glyco)proteins and glycolipids from the
plasma membrane to the Golgi complex and endoplasmic reticulum (17).
The best described of these systems is responsible for the retrieval of
proteins such as furin and TGN38 from the plasmalemma to the
trans-Golgi network (TGN), where they
reside. These proteins are internalized at the plasma membrane via
clathrin-coated vesicles and are targeted to the TGN by specific
domains of their cytoplasmic tail, which have been defined by
mutagenesis studies (13, 14, 36). Like the endocytic pathway, this
retrograde route requires luminal acidification for appropriate
intracellular traffic. Chapman and Munro (5) showed that delivery of
internalized furin and TGN38 to the TGN can be prevented by the weak
base chloroquine.
Endogenous retrograde pathways are also utilized by a number of
bacterial toxins to reach their target compartment within the cell.
Several such toxins share a double-moiety structure: a homopentameric B
subunit that binds to cell surface receptors (generally glycolipids)
(7) and a toxic A subunit that exerts its activity in the cytosol.
After binding to their receptors at the cell surface, these toxins are
internalized and retrogradely targeted through the TGN to the Golgi
complex and in some cases to the endoplasmic reticulum. The toxic
subunit is believed to enter the cytosol by crossing the membrane of
these secretory compartments (27). Such is the case for
Vibrio cholerae toxin (CT), which
specifically binds to the glycolipid monoganglioside 1 (GM1) at the plasma membrane.
After reaching the cytosol, the toxic
A1 fragment of the A subunit of CT
catalyzes ADP ribosylation of the
-subunit of heterotrimeric
Gs proteins, leading to persistent activation of adenylate cyclase (18, 34). Other members of this family
are Shiga toxin, the infectious agent in dysentery, which is produced
by Shigella dysenteriae (28), and
verotoxin (VT). VT, which is synthesized by enterohemorrhagic strains
of Escherichia coli, is associated
with human vascular diseases such as hemorrhagic colitis and hemolytic
uremic syndrome (20). In spite of their different origins, VT and Shiga
toxin have a high degree of structural homology and share the same
receptor, the glycolipid globotriaosyl ceramide
(Gb3). These two toxins also utilize the same intoxication mechanism: after these toxins reach the
endoplasmic reticulum, their A subunits presumably translocate to the
cytosol where they inactivate the 60S ribosomal subunits by
depurination of a specific adenine residue, causing inhibition of
protein synthesis (32).
The mechanism(s) whereby such bacterial toxins gain access to
the Golgi complex and endoplasmic reticulum has not been
adequately elucidated. CT has been reported to enter
the cells through caveolae (22). In contrast, the process underlying VT
internalization is not well defined. Initial studies in Daudi cells
showed that monodansyl cadaverine prevents endocytosis of the B
subunit of verotoxin 1 (VT1B) (15). This alkyl amine
competitively inhibits cytosolic transglutaminase activity, an enzyme
involved in the cross-linking of ligand-bound cell surface receptors
clustered within clathrin-coated pits. The subsequent steps in the
retrograde transport of VT, CT, and Shiga toxin to the Golgi complex
have not been explored. In particular, it is unclear whether the toxins utilize the same pathway that mediates retrieval of furin and TGN38 to
the Golgi complex.
In the present work, we characterized the internalization and
retrograde transport of VT1B and of the B subunit of CT (CTB) in Vero
cells. To minimize toxicity, we omitted the A subunits and utilized
recombinant B subunits, which dictate the receptor affinity and
targeting of the toxins. The results obtained suggest that VT1B can
enter the cells by both clathrin-dependent and -independent routes.
More importantly, the targeting of VT1B and CTB to the Golgi complex
was found to be mediated by a common retrograde pathway, which is
distinct from the furin and TGN38 retrieval system.
 |
MATERIALS AND METHODS |
Materials.
Texas red-labeled transferrin and rhodamine-labeled phalloidin were
purchased from Molecular Probes (Eugene, OR). Indocarbocyanine (Cy3)-labeled donkey anti-mouse antibodies were from
Jackson Immuno Research Laboratories (West Grove, PA). Fluorescein
isothiocyanate (FITC)-labeled CTB, filipin, and cytochalasin D were
purchased from Sigma Chemical (St. Louis, MO). Concanamycin A was from
Kamiya Biochemical (Thousand Oaks, CA), and monodansyl cadaverine was from Fluka Chemie (Buchs, Switzerland).
125I-labeled goat anti-human
immunoglobulin G (IgG) antibody was from ICN (Costa Mesa, CA). The
monoclonal antibody 10E6 was the kind gift of Dr. W. Brown (Cornell
University, Ithaca, NY).
Recombinant VT1B was purified by affinity chromatography and labeled
with fluorescein by the addition of FITC [1:1 (wt/wt) ratio] in 0.5 M
Na2CO3-NaHCO3
(pH 9.5). The mixture was gently rotated for 1-2 h at room
temperature, after which free FITC was removed by dialysis. Rhodamine
isothiocyanate (RITC)-labeled VT1B was similarly prepared.
Cell culture and incubation with toxins.
Vero cells obtained from the American Type Culture Collection
(Rockville, MD) were cultured at 37°C in minimal essential medium (MEM; GIBCO, Grand Island, NY) containing 5% heat-inactivated fetal
bovine serum (Cansera International, Rexdale, ON, Canada), 0.1%
glutamine, vitamins, and 1% penicillin-streptomycin (GIBCO) under 5%
CO2.
Vero cells, grown to near confluence on 18-mm-diameter glass
coverslips, were washed three times in cold Dulbecco's modified buffered saline solution (PBS; Pierce, Rockford, IL) containing 1 mM
CaCl2 and 1 mM
MgCl2 (pH 7.4). To monitor the
distribution of FITC-VT1B or FITC-CTB, the cells were exposed to 10 µg/ml of the appropriate toxin in PBS for 1 h at 4°C to promote
binding to the plasmalemmal receptors without endocytosis. After cells were washed twice with PBS, internalization was initiated by incubating the cells at 37°C for the specified periods.
Chinese hamster ovary cells stably transfected with a furin-IgG
chimeric construct were kindly provided by Drs. K. Teter and H. P. Moore, Department of Cell Biology, University of California at Berkeley
(Berkeley, CA).
Protocols for inhibition of endocytosis.
Four different protocols were used to inhibit clathrin-mediated
internalization. 1) The first protocol was
monodansyl cadaverine treatment: cells were incubated with the
appropriate toxin, as described above, in the presence of 500 µM
monodansyl cadaverine. 2) The second protocol
was K+ depletion: cells were
subjected to a hypotonic shock by preincubation in 50% diluted culture
medium for 5 min at 37°C. The cells were then washed with PBS and
incubated in K+-free medium
[140 mM NaCl, 10 mM
N-methyl-D-glucamine
chloride, 1 mM MgCl2, 1 mM
CaCl2, 20 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 5.5 mM glucose, and 1% bovine serum albumin (BSA; Boehringer Mannheim), pH 7.4] for 10 min at 37°C. Labeling
with the appropriate toxin was performed in the
K+-free medium.
3) The third protocol was hypertonic
treatment: cells were preincubated in hypertonic medium (in mM: 350 NaCl, 10 KCl, 1 MgCl2, 1 CaCl2, 20 HEPES, and 5.5 glucose
and 1% BSA, pH 7.4) for 20 min at 37°C. Labeling with the
appropriate toxin was also performed in the hypertonic medium.
4) The fourth protocol was
cytoplasmic acidification: cells were preincubated in growth medium
supplemented with 5 mM acetic acid (pH 5.0) for 5 min at 37°C. This
condition was maintained throughout the labeling of the cells with the
appropriate toxin.
Internalization via caveolae was prevented by treatment with 5 µg/ml
filipin immediately before and during incubation with the toxins.
Immunocytochemistry and fluorescence microscopy.
Cells were fixed in 4% paraformaldehyde in PBS at room temperature for
30 min, washed with 100 mM glycine, and permeabilized with 0.1% Triton
X-100 in PBS. To label the cis-Golgi
cisternae, fixed and permeabilized cells were then blocked with 5%
donkey serum in PBS containing 0.1% BSA and 0.1% Triton X-100 for 20 min at room temperature, washed three times with PBS, and incubated with the monoclonal antibody 10E6 (1:200 dilution in PBS containing 0.1% BSA and 0.1% Triton X-100) for 2 h at room temperature. Samples were subsequently washed three times in PBS containing 0.1% BSA and
incubated with Cy3-labeled donkey anti-mouse antibody (1:500 dilution
in PBS containing 0.1% BSA and 0.1% Triton X-100) for 1 h at room
temperature. After samples were washed three times with PBS-0.1% BSA,
samples were treated with Slow Fade (Molecular Probes) and mounted.
Control experiments were performed, omitting the primary antibody.
F-actin was stained by incubating the fixed and permeabilized cells in
the presence of rhodamine-phalloidin (10 U/ml) for 30 min at room
temperature.
Analysis of the samples was performed using the ×100 objective of
either a Leica TCS4D laser confocal microscope or a Leica DM1RB
fluorescence microscope (Heidelberg, Germany) equipped with a Micromax
cooled charge-coupled device (CCD) camera (Princeton Instruments),
operated from a Dell computer using Winview software (Princeton
Instruments). Digitized images were cropped in Adobe Photoshop and
imported to Adobe Illustrator (Adobe Systems, Mountain View, CA) for
assembly and labeling.
Quantitative analysis of toxin binding.
To analyze the interaction between toxins, cells were incubated with 10 µg/ml of unlabeled VT1B or 10 µg/ml of unlabeled CTB for 1 h at
4°C, washed, and warmed to 37°C for 15 min to allow internalization. The samples were then incubated with either FITC-CTB or FITC-VT1B, respectively, for 1 h at 4°C and washed. After the unbound toxin was washed, the cells were lysed by incubation in 50 mM
tris(hydroxymethyl)aminomethane and 1% Nonidet P-40 (pH 8) for 5 min.
After a thorough mixing, debris were sedimented in an Eppendorf 5415 microcentrifuge and the fluorescence intensity of the supernatants was
quantified in a Perkin Elmer 650-40 fluorescence spectrophotometer.
Background fluorescence was determined using nonlabeled cells and
subtracted from all other determinations. The results were expressed as
the ratio of the fluorescence intensity of toxin-pretreated and control
(nonpretreated) cells.
Fluorescence ratio and Nomarski imaging.
Simultaneous imaging of fluorescence and of cell morphology was
performed using an inverted microscope (Axiovert 135; Zeiss, Oberkochen, Germany) equipped with epifluorescence optics. Excitation at 440 and 490 nm was provided by a xenon arc lamp via a
computer-controlled shutter and filter wheel assembly (Sutter
Instruments, Novato, CA), whereas continuous 620-nm illumination was
achieved by filtering the transmitted incandescent source. The
excitation light was attenuated by a neutral density filter and
reflected to the cells by a dichroic mirror (510 nm), while the emitted
fluorescence (>510 nm) and the transmitted red light (>620 nm) were
separated by an emission dichroic mirror (580 nm). The red light was
directed to a video camera, allowing continuous visualization of the
cells, while the fluorescent light was directed onto a 542 band-pass 62-nm filter and imaged with a cooled CCD camera
(Princeton Instruments). Control of image acquisition was achieved with
Metafluor software (Universal Imaging, West Chester, PA), operating on
a pentium Dell computer (Dell, Toronto, ON, Canada).
For imaging experiments, the cells were grown on 25-mm-diameter glass
coverslips (Thomas Scientific, Swedesboro, NJ) that were inserted into
a Leiden coverslip dish (Medical Systems, Greenvale, NY), which was in
turn placed into a thermostatted perfusion chamber (open perfusion
Micro-Incubator, Medical Systems). Regions of interest were selected
for measurement by the imaging system. Background fluorescence was
subtracted for each wavelength within each experiment. At the end of
each experiment, a calibration curve of fluorescence vs. pH was
obtained in situ by sequentially perfusing the cells with KCl-rich
medium containing (in mM) 125 KCl, 20 NaCl, 10 HEPES, 10 2-(N-morpholino)ethanesulfonic acid, 0.5 CaCl2, and 0.5 MgCl2 in the presence of 5 µM
nigericin and buffered to pH values ranging from 5.5 to 7.5. The
theoretical basis of this method was described earlier (15).
Approximately 3 min were allowed for equilibration at each pH.
All confocal and ratio images are representative of at least three
separate experiments.
VT1B uptake determinations.
Cells were incubated with 10 µg/ml VT1B for 1 h at 4°C, followed
by a chase at 37°C for the specified periods of time. Samples were
fixed as above and then blocked with 5% donkey serum in PBS containing
0.1% BSA for 20 min at room temperature. After cells were washed three
times with PBS, cells were incubated with monoclonal anti-VT1B antibody
(3) (1:500 dilution in PBS containing 0.1% BSA) for 1 h at room
temperature. Samples were subsequently washed three times in PBS
containing 0.1% BSA and incubated with
125I-labeled goat anti-mouse
antibody (0.1 µCi/ml in PBS with 0.1% BSA) for 1 h at room
temperature. After three more washes, the cells were scraped and
transferred to counting vials. Radioactivity was quantified using a
1282 Compu-Gamma counter (LKB Wallac, Turku, Finland). Background
radioactivity, determined with the omission of the primary antibody,
was subtracted from all other determinations.
 |
RESULTS |
Time course of VT1B and CTB internalization.
The available evidence suggests that VT1B and CTB enter the cells via
distinct mechanisms (see introduction). Their rate of internalization
and subcellular distribution would therefore be expected to differ.
This notion was tested by coincubating Vero cells with the two toxins
and monitoring their fate by confocal fluorescence microscopy. The cell
surface was labeled with a mixture of RITC-VT1B and FITC-CTB at 4°C
(Fig. 1,
A-C),
the cells were then warmed to 37°C, and samples were taken at
increasing times (Fig. 1,
D-O).
The toxins, which are initially seen lining the surface membrane (Fig.
1,
A-C),
subsequently assume a punctate distribution and eventually cluster in a
tight juxtanuclear complex, likely the Golgi complex.
Overlay of the red and green fluorescence (Fig. 1)
revealed that the distribution of the two toxins is very similar
throughout, suggesting that VT1B and CTB utilize a common pathway.

View larger version (59K):
[in this window]
[in a new window]
|
Fig. 1.
Time course of B subunit of verotoxin 1 (VT1B) and B subunit of CT
(CTB) internalization. Vero cells were incubated with rhodamine
isothiocyanate (RITC) VT1B and fluorescein isothiocyanate (FITC) CTB
immediately (10 µg/ml each) for 1 h at 4°C and either fixed
immediately (A-C) or incubated
at 37°C for the indicated periods of time
(D-O). Left
column: distribution of VT1B. Middle
column: distribution of CTB. Right
column: areas of VT1B and CTB colocalization (overlay).
Results are representative of 3 similar experiments.
|
|
Several lines of evidence indicate that the morphological
redistribution of the toxins is associated with their internalization. First, although VT1B bound to the surface of the cells at 4°C is
readily accessible to a specific antibody added externally (Fig.
2A),
incubation of the cells for 1 h at 37°C renders the toxin
inaccessible to the antibody (Fig.
2B). In fact, the course of
disappearance of VT1B from the surface, assessed by binding of
anti-VT1B antibody followed by a radiolabeled secondary antibody (Fig.
2D), corresponds well to the
redistribution monitored morphologically (Fig. 1). It is noteworthy
that, once internalized, VT1B seemingly does not recycle back to the
surface membrane. This was concluded from the observation that no
labeling was observed even when the anti-VT1B antibody was added to the
intact cells for 1 h at 37°C after the toxin was internalized (Fig.
2C). Significant recycling of the
toxin to the surface would have resulted in progressive recruitment of
extracellular antibody.

View larger version (54K):
[in this window]
[in a new window]
|
Fig. 2.
Internalization of VT1B. A: cells were
exposed to VT1B for 1 h at 4°C and immediately fixed and labeled
sequentially with anti-VT1B monoclonal (primary) antibody (Ab) followed
by indocarbocyanine-labeled goat anti-mouse (secondary) Ab.
B: cells were exposed to VT1B at
4°C and then allowed to internalize the toxin for 1 h at 37°C.
After fixation, cells were incubated with primary and secondary Abs as
in A.
C: cells were exposed to VT1B at
4°C and then allowed to internalize the toxin for 1 h at 37°C.
Intact cells were next exposed to the primary Ab at 37°C for an
additional 1 h. Finally, cells were fixed and labeled with secondary
Abs as in A.
A-C
are representative of 3 similar experiments.
D: cells grown on 6-well plates were
preincubated with VT1B for 1 h at 4°C, followed by an incubation
for the specified periods of time at 37°C (abscissa). Samples were
then fixed and incubated sequentially with primary Ab, followed by
125I-labeled goat-anti-mouse Ab,
as described in MATERIALS AND METHODS.
Radioactivity associated with confluent cells on a well is plotted vs.
time. Data are means of 2 determinations. cpm, Counts per minute.
|
|
The occurrence of internalization is also supported by the observation
that, shortly after warming up, both CTB and VT1B colocalize extensively with transferrin receptors, the conventional markers of
early and recycling endosomes (Fig. 3).
Together, these observations suggest that VT1B and CTB enter the cells
by similar pathways and, within the period studied, do not recycle
detectably back to the surface membrane.

View larger version (88K):
[in this window]
[in a new window]
|
Fig. 3.
Endosomal transit of VT1B and CTB. Vero cells were incubated with 5 µg/ml of Texas red-labeled transferrin and 10 µg/ml of either
FITC-VT1B or FITC-CTB for 1 h at 4°C. Cells were then washed, and
internalization was allowed to occur in MEM for 15 min at 37°C.
Cells were fixed, mounted, and visualized by epifluorescence
microscopy. A and
C: localization of transferrin.
B: localization of VT1B in the same
cells shown in A.
D: localization of CTB in the same
cells shown in C. Results are
representative of 3 similar experiments.
|
|
Clathrin dependence of VT1B internalization.
The similarities in the behavior of the two toxins contrast with the
notion that they enter via different mechanisms. To better define the
pathway utilized by VT, we studied the effects of three protocols
routinely used to inhibit clathrin-mediated endocytosis, namely,
K+ depletion, hypertonic
treatment, and cytoplasmic acidification, on the uptake of FITC-VT1B by
Vero cells. Whereas K+ depletion
and hypertonic treatment inhibit internalization by dispersing
membrane-associated clathrin lattices, cytoplasmic acidification
prevents the budding of clathrin-coated vesicles from the plasma
membrane (11). To test the efficiency of these treatments in Vero
cells, we assessed the uptake of transferrin, which is typically
internalized through clathrin-coated pits and vesicles. Cells were
preincubated in the presence of Texas red-labeled transferrin for 1 h
at 4°C, washed, and incubated at 37°C for 20 min. The
distribution of the fluorophore was then determined by confocal
microscopy, and representative x vs.
z reconstructions are shown in Fig.
4. Whereas transferrin was normally
internalized in untreated cells (Fig.
4A),
K+-depleted cells displayed only
labeling on the surface (Fig. 4B). Virtually identical results were obtained in cells treated
hypertonically or with acidic solutions (Fig. 4,
C and
D).

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of K+ depletion, hypertonic
treatment, and cytoplasmic acidification on transferrin endocytosis.
Vero cells were either untreated
(A), depleted of intracellular
K+ as described in
MATERIALS AND METHODS
(B), preincubated in hypertonic
medium for 20 min at 37°C (C),
or exposed to acidifying medium for 5 min at 37°C
(D). Next, cells were incubated with
Texas red-labeled transferrin (5 µg/ml) for 1 h at 4°C in the
appropriate solution. Cells were then washed, and internalization was
allowed to occur in the appropriate media for 15 min at 37°C. Cells
were fixed, mounted, and visualized by confocal microscopy.
Representative x vs.
z (cross-sectional) reconstructions
are shown. Results are representative of 3 similar experiments. Bar = 5 µm.
|
|
Having confirmed the effectiveness of the treatments used for
inhibition of clathrin-mediated endocytosis, we proceeded to determine
the role of clathrin in VT1B internalization. As shown above and
reported earlier, when allowed to internalize for 1 h, VT1B accumulates
in a juxtanuclear location that corresponds to the Golgi complex (Fig.
5A).
Figure 5B shows that
K+ depletion had no noticeable
effect on the distribution of VT1B. Similarly, near normal
internalization was observed in cells incubated in hypertonic medium
(Fig. 5C). These results imply that
VT1B can be internalized and reach the Golgi apparatus by
clathrin-independent means. In contrast to the effects of
K+ depletion and hypertonicity,
the juxtanuclear accumulation of VT1B was markedly inhibited by
cytosolic acidification (Fig. 5D). A
substantial portion of the toxin remained on the cell surface, whereas
a fraction was detected in small intracellular vesicles. This implies
that the harsher acidic treatment is less specific, likely affecting
processes other than clathrin-coated vesicle formation.

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 5.
Role of clathrin-mediated endocytosis in VT1B internalization. Vero
cells were either untreated (A),
depleted of intracellular K+ as
described in MATERIALS AND METHODS
(B), preincubated in hypertonic
medium for 20 min at 37°C (C),
or exposed to acidifying medium for 5 min at 37°C
(D). Next, cells were incubated with
FITC-VT1B (10 µg/ml) for 1 h at 4°C in the appropriate solution.
Cells were then washed, and internalization was allowed to occur in the
appropriate media for 1 more h at 37°C. Cells were fixed, mounted,
and visualized under confocal microscopy. Representative
x vs.
y scans are shown at
top and
x vs.
z (cross-sectional) reconstructions
are shown for each condition at
bottom. In this and Figs. 6, 7, and
10, the cross-sectional views were obtained from cells other than those
shown in the x vs.
y scans. Results are representative of
3 similar experiments.
|
|
Prima facie, there is an apparent discrepancy between our
observations and the results reported in Daudi cells, in which
monodansyl cadaverine prevented VT1B internalization. In an attempt to
reconcile these results, we tested the effect of this cadaverine
derivative in Vero cells. As shown in Fig.
6A,
monodansyl cadaverine effectively inhibited transferrin uptake in
Vero cells. However, the internalization of VT1B proceeded
normally in the presence of the transglutaminase inhibitor (Fig.
6B), confirming internalization by a
clathrin-independent route.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 6.
Effects of monodansyl cadaverine on the internalization of transferrin,
VT1B, and CTB. Vero cells were preincubated in medium containing 500 µM monodansyl cadaverine for 30 min at 4°C. Next, cells were
incubated with either 5 µg/ml Texas red-labeled transferrin
(A), 10 µg/ml FITC-VT1B
(B), or 10 µg/ml FITC-CTB
(C) for 1 h at 4°C in solution
containing monodansyl cadaverine. Cells were then washed and incubated
in monodansyl cadaverine-containing medium for 1 more h at 37°C.
Cells were finally fixed, mounted, and visualized by confocal
microscopy. A: representative
x vs.
z (cross-sectional) reconstructions.
B and
C: representative
x vs.
y scans
(B and
C) are shown at
top and
x vs.
z are shown at
bottom. Results are representative of
3 similar experiments.
|
|
As shown in Fig.
7A, CTB
also accumulates in the Golgi complex of Vero cells. The
internalization of this toxin by A-431 cells is believed to be mediated
by caveolae (22). This is likely also to be the case in Vero cells,
inasmuch as the uptake and juxtanuclear accumulation of CTB were
unaffected by K+ depletion,
hypertonicity, or monodansyl cadaverine (Fig. 7,
B and
C, and Fig.
6C), as was the case for VT1B. It is
noteworthy that CTB uptake was markedly inhibited by acid
loading. This observation supports the earlier notion that cytosolic
acidification exerts multiple effects, inhibiting both
clathrin-dependent and -independent processes.

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 7.
Role of clathrin-mediated endocytosis in CTB internalization. Vero
cells were either untreated (A),
depleted of intracellular K+ as
described in MATERIALS AND METHODS
(B), preincubated in hypertonic
medium for 20 min at 37°C (C),
or exposed to acidifying medium for 5 min at 37°C
(D). Next, the cells were incubated
with FITC-CTB (10 µg/ml) for 1 h at 4°C in the appropriate
solution. Cells were then washed, and internalization was allowed to
occur in the appropriate media for 1 more h at 37°C. Cells were
fixed, mounted, and visualized by confocal microscopy. Representative
x vs.
y scans are shown at
top and
x vs.
z (cross-sectional) reconstructions
are shown for each condition at
bottom. Results are representative of
3 similar experiments.
|
|
Effect of cytochalasin B on VT1B and CTB internalization and
retrograde transport.
Clathrin-independent internalization of plasmalemmal components can
occur by macropinocytosis, which is observed at sites of active
ruffling. Unlike clathrin-dependent vesicle formation, macropinocytosis requires assembly of filamentous (F) actin and is
therefore sensitive to the cytochalasins. To test the possible involvement of macropinosomes in VT1B uptake, Vero cells were pretreated with 5 µM cytochalasin B, which binds to the barbed (plus)
end of F-actin, inhibiting its polymerization. The effectiveness of
this drug was ascertained by staining F-actin with
rhodamine-phalloidin, as illustrated in Figs.
8, A and
C, and 9,
A and
C. Cytochalasin virtually eliminated
the stress fibers and decreased the overall content of F-actin, which
was largely clustered in irregular aggregates (cf.
A and
C in Figs. 8 and 9). Figure 8 also
shows that VT1B was effectively internalized in cytochalasin-treated
cells. It is noteworthy that the morphology of the intracellular
compartment where VT1B was accumulated was altered by F-actin
disruption. However, this does not reflect a change in the targeting of
the toxin but rather an alteration in Golgi morphology. This was
determined by comparing the distribution of the toxin with that of a
cis-Golgi marker, identified by
antibody 10E6 (Fig. 8, E and
F). Similar results were obtained
using CTB (Fig. 9). These findings indicate that neither
macropinocytosis nor other F-actin-dependent processes are important in
VT1B internalization. Moreover, they imply that actin polymerization is
not required for the retrograde targeting of either VT1B or CTB to the
Golgi.

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 8.
Effect of cytochalasin B on the internalization and targeting of VT1B.
Vero cells were allowed to internalize FITC-VT1B in the absence
(A and
B) or presence
(C-F) of cytochalasin D (Cyto
D; 5 µM). A and
C: actin filaments were stained with
rhodamine-phalloidin. B,
D, and
F: VT1B fluorescence.
E: indirect immunofluorescence using
Ab 10E6, directed to an epitope in the
cis-Golgi. Results are representative
of 3 similar experiments.
|
|

View larger version (43K):
[in this window]
[in a new window]
|
Fig. 9.
Effect of cytochalasin B on the internalization and targeting of CTB.
Vero cells were allowed to internalize FITC-CTB in the absence
(A and
B) or presence
(C-F) of cytochalasin D (5 µM). A and
C: actin filaments were stained with
rhodamine-phalloidin. B,
D, and
F: CTB fluorescence.
E: indirect immunofluorescence using
Ab 10E6, directed to an epitope in the
cis-Golgi. Results are representative
of 3 similar experiments.
|
|
Involvement of caveolae in VT1B internalization.
Clathrin-independent internalization can also occur through caveolae,
which are 50- to 60-nm plasma membrane invaginations with a
characteristic flask shape. These structures are enriched in
glycosphingolipids and cholesterol and contain caveolin, a 21-kDa
cholesterol-binding protein that cycles between the plasma membrane and
the Golgi complex (23). Considering the similar subcellular
distribution of VT1B and CTB and their common resistance to antagonists
of clathrin-mediated uptake, it appeared likely that VT1B also utilizes
caveolae for internalization. This notion was tested with filipin, a
cholesterol-binding drug that causes disassembly of caveolae. In cells
treated with filipin (5 µg/ml), the internalization of VT1B was
partially inhibited (Fig.
10A). The toxin displayed a punctate distribution, with moderate perinuclear accumulation.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 10.
Role of caveolae in VT1B internalization.
A: Vero cells were preincubated with
FITC-VT1B for 1 h at 4°C in the presence of 5 µg/ml of filipin
and then were incubated for another hour at 37°C in
filipin-containing solution. B: cells
were preincubated with hypertonic medium as in Fig.
1C and then labeled at 4°C with
FITC-VT1B in the same medium with added filipin. These conditions were
maintained throughout the internalization chase (1 h at 37°C).
Representative x vs.
y scans and
x vs.
z reconstructions are shown for each
condition in top and
bottom, respectively. Results are
representative of 3 similar experiments.
|
|
This finding could reflect incomplete inhibition of caveolae formation
or the presence of an alternate internalization pathway. The latter
possibility was analyzed by combining the use of filipin with
hypertonic treatment, a mild procedure for inhibition of clathrin-mediated endocytosis. As illustrated in Fig.
10B, the inhibition of VT1B uptake was
virtually complete under these conditions. This observation implies
that VT1B can be endocytosed via clathrin-dependent as well as
clathrin-independent routes, likely caveolae. The failure to notice
significant inhibition when inhibiting only the former suggests that
caveolae are the predominant pathway and/or that one route is
upregulated when the other is inhibited.
pH dependence of VT1B and CTB retrograde transport.
The retrograde transport of furin and TGN38 from the plasma membrane to
the Golgi complex requires intraorganellar acidification (see
introduction). To determine whether toxins such as VTB utilize a
similar pH-dependent pathway, we monitored their distribution under
conditions expected to dissipate the electrochemical
H+ gradient of endomembrane
compartments. The luminal pH of endosomes and of components of the
Golgi apparatus is maintained as acidic by vacuolar
H+ pumps, which are selectively
inhibited by macrolide antibiotics such as the bafilomycins and
concanamycins. Cells were therefore treated with concanamycin A (100 nM) to inhibit active pumping. To ensure elimination of preexisting
H+ gradients, the cells were
additionally exposed to the weak base chloroquine (50 µM). The
dissipation of the organellar acidification was initially verified by
measuring the pH of endosomes and Golgi complex by imaging the emission
of FITC-labeled CTB, a pH-sensitive fluorescent probe. Cells were
preincubated with FITC-CTB for 1 h at 4°C, washed, and then
incubated for an additional 20 min at 37°C to allow
internalization. At this time, the probe is localized largely in
endosomes, although some of the juxtanuclear toxin may have entered the
TGN (Fig.
11A).
Endosomal pH was then monitored by quantifying the fluorescence with
excitation at 440 and 490 nm (see MATERIALS AND
METHODS). As illustrated in Fig.
11B, the pH in otherwise untreated
cells averaged 6.5 ± 0.03 (mean ± SE of 30 determinations from
3 experiments), in the range reported for early and recycling endosomes
(8). The combined treatment with concanamycin and chloroquine elevated
pH to 7.1 ± 0.09 (mean ± SE of 30 determinations).
Alkalinization was similar in the juxtanuclear and submembranous
endosomes.

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 11.
Effect of concanamycin and chloroquine on the pH of endomembrane
compartments. A: Vero cells were
preincubated with FITC-CTB for 1 h at 4°C, washed, and then
incubated for 20 more min at 37°C to allow internalization of the
toxin. A representative fluorescence micrograph, obtained using the
imaging setup described in MATERIALS AND
METHODS, is illustrated.
B: pH of the endomembrane compartments
labeled with FITC-CTB as in A was
measured by fluorescence ratio imaging in otherwise untreated cells
(control) and in cells incubated with 100 nM concanamycin (CCM) and 50 µM chloroquine (CLQ) during the last 30 min of the incubation at
4°C and throughout the incubation at 37°C. Data are means ± SE of 30 determinations.
|
|
Having demonstrated the effectiveness of the pH dissipation protocol,
we proceeded to test the pH dependence of VT1B retrograde transport.
Cells were treated with concanamycin and chloroquine during the last
0.5 h of the preincubation at 4°C with the toxin and throughout the
internalization period at 37°C. Figure
12 shows that conditions described above
to eliminate intraorganellar pH gradients had little effect on VT1B
internalization. The targeting of the toxin to the Golgi complex was
confirmed by dual labeling with the marker antibody 10E6 (cf. Fig. 12,
A-D).
These observations indicate that transport of VT1B from the membrane to
the Golgi complex does not require endosomal acidification. Retention
of the toxin within the Golgi complex is similarly independent of the
activity of the V-ATPase. This was shown in the experiments of Fig. 12,
E-F.
VT1B was allowed to reach the Golgi under normal conditions, and the pH
gradient was then dissipated as above. The distribution of fluorescence
was analyzed following an additional 2-h incubation in the presence of
concanamycin A and chloroquine. Such a long incubation with the
inhibitors resulted in visible swelling of the Golgi, yet the toxin was
retained within this compartment. These findings indicate that the
toxin does not cycle between the Golgi and other compartments over
the time course analyzed or that such recycling occurs in a
pH-independent manner. The pH dependence of the retrograde targeting
and retention of CTB was analyzed similarly. As summarized in Fig.
13, CTB was directed to the Golgi complex
even after dissipation of intraorganellar acidification and was
retained therein for at least 2 h in the absence of a pH gradient.

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 12.
pH dependence of VT1B internalization. Vero cells were labeled with
FITC-VT1B in the cold and then incubated in the absence
(A and
B) or presence
(C-F) of concanamycin (100 nM)
and chloroquine (50 µM) as indicated. At the end of the experiment,
cells were fixed, permeabilized, and labeled by indirect
immunofluorescence using Ab 10E6. Representative
x vs.
y confocal scans are illustrated.
Results are representative of 3 similar experiments.
|
|

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 13.
pH dependence of CTB internalization. Vero cells were labeled with
FITC-CTB in the cold and then incubated in the absence
(A and
B) or presence
(C-F) of concanamycin (100 nM)
and chloroquine (50 µM) as indicated. At the end of the experiment,
cells were fixed, permeabilized, and labeled by indirect
immunofluorescence using Ab 10E6. Representative
x vs.
y confocal scans are illustrated.
Results are representative of 3 similar experiments.
|
|
It was important to ascertain that, under the conditions used,
targeting of resident proteins to the TGN was impaired, as had been
reported (5). To this end, we used cells stably transfected with a
chimeric protein consisting of the cytosolic and transmembrane domains
of furin, which dictate TGN localization, attached to an extracellular
epitope that was readily detectable immunologically (a portion of human
IgG). As shown in Fig.
14A,
such a chimeric protein concentrates in the juxtanuclear location
anticipated for the TGN. More importantly, treatment with the
combination of concanamycin (100 nM) and chloroquine (50 µM) resulted
in dispersal of furin-IgG in a diffuse vesicular pattern (Fig.
14B), consistent with earlier
observations (5). Under comparable conditions, CTB targeted normally to
the Golgi complex, where it retained a compact distribution (cf. Fig.
14, C and
D). These observations highlight the
differential behavior of the retrograde pathways of proteins and
lipids.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 14.
pH dependence of furin and CTB internalization.
A and
B: Chinese hamster ovary (CHO) cells
expressing immunoglobulin G (IgG)-furin chimeras were incubated in the
absence (A) or presence
(B) of concanamycin (100 nM) and
chloroquine (50 µM) for 1 h 30 min at 37°C. Fixed and
permeabilized samples were then labeled by indirect immunofluorescence
using anti-human IgG antibodies. C and
D: CHO cells were preincubated with
FITC-CTB for 1 h at 4°C in the absence
(C) or presence
(D) of concanamycin (100 nM) and
chloroquine (50 µM), followed by incubation for an additional 1 h at
37°C under the same conditions. Results are representative of 2 similar experiments.
|
|
Are VT1B and CTB receptors associated?
Association among lipids with similar functional and/or
structural characteristics in microdomains often called "rafts"
has been shown or postulated to exist in different systems. Such
lipid-rich microdomains are seemingly involved in sorting and targeting
processes (4). Because the receptors for VT1B and CTB (namely,
Gb3 and GM1) are both glycolipids, we
considered the possibility that they are physically associated in
macromolecular complexes, possibly rafts. We reasoned that, if stable
association exists, internalization of one of the glycolipids would
drive the uptake of the other and vice versa. This was tested
experimentally by inducing the uptake of one of the lipid receptors by
addition of its unlabeled cognate toxin and measuring the degree of
surface exposure of the other with the respective tagged toxin. To
confirm that a sizable fraction of the first lipid was internalized, we
initially performed experiments measuring the effect of each toxin on
the ability of a second pulse of the same toxin to bind to the surface. Pretreatment with unlabeled CTB was followed by incubation for 15 min
at 37°C, to allow internalization, and then by exposure to
FITC-CTB, which was finally quantified fluorometrically (see MATERIALS AND METHODS). As shown in
Table 1, upwards of 60% of the
GM1 became inaccessible after
incubation with 10 µg/ml CTB. Similarly, nearly 55% of the
Gb3 was inaccessible after
treatment with 10 µg/ml of VT1B. In contrast, pretreatment with VT1B
had no detectable effect on the amount of
GM1 available at the plasma membrane and CTB only modestly decreased the surface-exposed
Gb3, as measured by VT1B binding.
These observations indicate that GM1 and
Gb3 are internalized independently
and not as part of tightly coupled macromolecular complexes.
 |
DISCUSSION |
Endogenous retrograde pathways directing molecules from the plasma
membrane to compartments of the secretory pathway can be subverted by
bacterial toxins, which travel through endosomes to the Golgi complex
and sometimes as far back as the endoplasmic reticulum and nuclear
envelope. Several lines of evidence indicate that such retrograde
translocation is essential for the toxins to exert their biological
effects. In particular, it has been shown that cells are protected
against the toxic effects of these bacterial polypeptides by brefeldin
A (30) and by overexpression of inactive mutants of Rab1, Sar1, and
Arf1 (33), which preclude retrograde transport between the Golgi and
the endoplasmic reticulum.
Although different aspects of the uptake of toxins have been
investigated (29, 31), the nature of the retrograde route mediating
their transport from the plasma membrane has not been elucidated. In
this report, we compared the internalization and targeting of VT1B and
CTB and showed that they share steps of a common retrograde pathway
that differs, at least in part, from the one employed by furin and
TGN38.
VT1B internalization.
VT1B appears to be internalized in Vero cells by two distinct pathways:
one that is clathrin dependent and one (or more) clathrin-independent route(s). Endocytosis was partially inhibited by filipin, suggesting the involvement of caveolae, whereas the combination of this drug with
hypertonicity resulted in complete inhibition, implying a contribution
from clathrin-coated vesicles. We were unable to determine whether both
routes are constitutively active or whether the inhibition of one of
them caused upregulation of the other. In addition, the specificity of
the pharmacological agents used may not be absolute, so that
alternative pathways cannot be ruled out. It is clear, however, that
macropinocytosis and other F-actin-dependent processes do not
contribute importantly to toxin uptake in Vero cells.
The involvement of caveolae in VT1B uptake is consistent with their
reported role in endocytosis of CT, tetanus, and heat-labile toxin (22,
24, 34). Importantly, a dual-internalization mechanism like the one
reported here was also observed for ricin, a toxin that binds terminal
galactose residues of glycolipids and glycoproteins (32, 33). In
contrast, endocytosis of Shiga toxin, which is almost identical to
VT1B, was believed to occur primarily via clathrin-coated pits (26,
28). The observed differences may be due to subtle structural
differences between these toxins or may reflect differences in the cell
types or experimental protocols used.
It is not clear how glycolipids localize to coated pits. It is
conceivable that they are specifically associated with transmembrane proteins that bear endocytic targeting sequences that bind to adaptor
complex 2. It is easier to rationalize the internalization of toxin
receptors via caveolae, where the glycolipids are originally abundant.
It remains to be determined if the glycolipids cycle constitutively
from the plasma membrane to the secretory pathway or if internalization
is triggered by binding to the toxins. The latter is pentavalent,
raising the possibility that cross-linking of multiple glycolipids and
their associated proteins and lipids induces active internalization.
Characterization of VT1B and CTB targeting to the Golgi complex.
Glycosphingolipids have been shown to form microdomains at the plasma
membrane and TGN. In polarized cells, TGN microdomains are involved in
sorting and specific targeting of secreted proteins through the
formation of rafts (4). However, the involvement of such rafts in
retrograde transport from the plasma membrane has not been determined.
Indeed, comparatively little is known about the lipid retrograde
transport pathways. Martin and Pagano (21) showed that, in fibroblasts,
glucosylceramide fluorescent analogs are internalized from the plasma
membrane by an ATP-independent and temperature-insensitive saturable
mechanism to the Golgi complex, where they can be further processed. In
contrast, internalization of fluorescent analogs of sphingomyelin is
temperature and energy dependent. These results suggest that retrograde
transport of glycolipids from the plasma membrane can be mediated by
both endocytic and nonendocytic pathways and show that these molecules
can be targeted to the Golgi complex. In the case of
Gb3 and
GM1, uptake is clearly temperature
sensitive and mediated by endosomes.
Ligands internalized through coated and uncoated pits have been found
within the same endosomal compartments, suggesting that these
structures are a meeting point for molecules entering by different
routes. The ligands and their receptors are then sorted and directed to
their final destinations. Delivery of early endosomal proteins to late
endosomes and lysosomes involves a step that is dependent on the
maintenance of an acidic luminal pH. The precise nature of the
pH-sensitive step is ill defined, but assembly of coat proteins before
budding of the endosomes has been proposed as the critical
acidification-dependent event (1). Importantly, retrieval of proteins
like furin and TGN38 from the membrane to the TGN is comparably pH
sensitive. It is therefore remarkable that retrograde targeting of
neither VT1B nor CTB was affected by a combination of chloroquine and
concanamycin. Parallel experiments confirmed that this combination of
agents neutralized the pH of the compartments involved in the
translocation of the toxins to the TGN (Fig. 11) and of the TGN itself
(data not shown) and altered the targeting of TGN-resident proteins
(Fig. 14). This result is consistent with observations made earlier
with other toxins. Treatment with either
NH4Cl, nigericin, or monensin was
unable to prevent the intoxication of Vero cells with Shiga toxin, a
process that requires retrograde transport (26). Similarly, in T84
cells, dissipation of the pH gradient with chloroquine, nigericin, or concanamycin did not alter internalization of CT or the subsequent signal transduction events (19). We therefore conclude that at least
two different retrograde pathways exist for delivery of plasmalemmal
macromolecules to the TGN: one that requires luminal acidification,
which is used by molecules such as furin and TGN38, and another one
that is pH independent and mediates retrograde transfer of glycolipids
and their ligands.
Finally, data were presented that suggested that internalization of
Gb3 and
GM1 occurs independently, arguing
against mediation by stable glycolipid rafts. It is possible that the
glycolipids exist within the membrane as monomeric entities or in
association with specific proteins. These may cycle constitutively
between the Golgi and the plasma membrane. However, no significant
forward transport was detected for hours after the lipid-toxin
complexes reached the Golgi region (e.g., see Figs.
2C, 12,
E and
F, and 13,
E and
F), arguing against constitutive
cycling. Alternatively, it is conceivable that addition of the
pentavalent toxins may induce cross-linking of the lipids and their
associated proteins, thereby signaling internalization. The detailed
mode of internalization remains to be defined.
 |
ACKNOWLEDGEMENTS |
We thank Drs. K. Teter and H. P. Moore (University of California at
Berkeley) for generously sharing their cells transfected with the
furin-IgG construct.
 |
FOOTNOTES |
This study was supported by the Canadian Cystic Fibrosis Foundation and
the Medical Research Council of Canada.
F. B. Schapiro is the recipient of a University of Toronto Connaught
Scholarship. S. Grinstein is an International Scholar of the Howard
Hughes Medical Institute.
Address for reprint requests: S. Grinstein, Division of Cell Biology,
Hospital for Sick Children, 555 Univ. Ave., Toronto, ON, Canada M5G
1X8.
Received 16 May 1997; accepted in final form 26 September 1997.
 |
REFERENCES |
1.
Aniento, F.,
F. Gu,
R. G. Parton,
and
J. Gruenberg.
An endosomal beta COP is involved in the pH-dependent formation of transport vesicles destined for late endosomes.
J. Cell Biol.
133:
29-41,
1996[Abstract].
2.
Boulanger, J.,
M. Huesca,
S. Arab,
and
C. A. Lingwood.
Universal method for the facile production of glycolipid/lipid matrices for the affinity purification of binding ligands.
Anal. Biochem.
217:
1-6,
1994[Medline].
3.
Boulanger, J.,
M. Patric,
C. Lingwood,
H. Law,
M. Roscoe,
and
M. Karmali.
Neutralization receptor-based immunoassay for detection of neutralizing antibodies to Escherichia coli verocytotoxin 1.
J. Clin. Microbiol.
28:
2830-2833,
1990[Medline].
4.
Brown, D. A.,
and
J. K. Rose.
Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface.
Cell
68:
533-544,
1992[Medline].
5.
Chapman, R. E.,
and
S. Munro.
Retrieval of TGN proteins from the cell surface requires endosomal acidification.
EMBO J.
13:
2305-2312,
1994[Abstract].
6.
Clague, M. J.,
S. Urbe,
F. Aniento,
and
J. Gruenberg.
Vacuolar ATPase activity is required for endosomal carrier vesicle formation.
J. Biol. Chem.
269:
21-24,
1994[Abstract/Free Full Text].
7.
Fishman, P. H.,
T. Pacuszka,
and
P. A. Orlandi.
Gangliosides as receptors for bacterial enterotoxins.
Adv. Lipid Res.
25:
165-187,
1993[Medline].
8.
Forgac, M.
Structure and function of vacuolar class of ATP-driven proton pumps.
Physiol. Rev.
69:
765-796,
1989[Free Full Text].
9.
Fuchs, R.,
P. Male,
and
I. Mellman.
Acidification and ion permeabilities of highly purified rat liver endosomes.
J. Biol. Chem.
264:
2212-2220,
1989[Abstract/Free Full Text].
10.
Geisow, M. J.
Fluorescein conjugates as indicators of subcellular pH. A critical evaluation.
Exp. Cell Res.
150:
29-35,
1984[Medline].
11.
Hansen, S. H.,
K. Sandvig,
and
B. van Deurs.
Clathrin and HA2 adaptors: effects of potassium depletion, hypertonic medium, and cytosol acidification.
J. Cell Biol.
121:
61-72,
1993[Abstract].
12.
Harvey, W. R.
Physiology of V-ATPases.
J. Exp. Biol.
172:
1-17,
1992[Medline].
13.
Humphrey, J. S.,
P. J. Peters,
L. C. Yuan,
and
J. S. Bonifacino.
Localization of TGN38 to the trans-Golgi network: involvement of a cytoplasmic tyrosine-containing sequence.
J. Cell Biol.
120:
1123-1135,
1993[Abstract].
14.
Jones, B. G.,
L. Thomas,
S. S. Molloy,
C. D. Thulin,
M. D. Fry,
K. A. Walsh,
and
G. Thomas.
Intracellular trafficking of furin is modulated by the phosphorylation state of a casein kinase II site in its cytoplasmic tail.
EMBO J.
14:
5869-5883,
1995[Abstract].
15.
Khine, A. A.,
and
C. A. Lingwood.
Capping and receptor-mediated endocytosis of cell-bound verotoxin (Shiga-like toxin). 1. Chemical identification of an amino acid in the B subunit necessary for efficient receptor glycolipid binding and cellular internalization.
J. Cell. Physiol.
161:
319-332,
1994[Medline].
16.
Kim, J. H.,
C. A. Lingwood,
D. B. Williams,
W. Furuya,
M. F. Manolson,
and
S. Grinstein.
Dynamic measurement of the pH of the Golgi complex in living cells using retrograde transport of the verotoxin receptor.
J. Cell Biol.
134:
1387-1399,
1996[Abstract].
17.
Koval, M.,
and
R. E. Pagano.
Lipid recycling between the plasma membrane and intracellular compartments: transport and metabolism of fluorescent sphingomyelin analogues in cultured fibroblasts.
J. Cell Biol.
108:
2169-2181,
1989[Abstract].
18.
Lencer, W. I.,
C. Constable,
S. Moe,
M. G. Jobbling,
H. M. Webb,
S. Ruston,
J. L. Madara,
T. R. Hirst,
and
R. K. Holmes.
Targeting of cholera toxin and Escherichia coli heat labile toxin in polarized epithelia: role of COOH-terminal KDEL.
J. Cell Biol.
131:
951-962,
1995[Abstract].
19.
Lencer, W. I.,
G. Strohmeier,
S. Moe,
S. L. Carlson,
C. T. Constable,
and
J. L. Madara.
Signal transduction by cholera toxin: processing in vesicular compartments does not require acidification.
Am. J. Physiol.
269 (Gastrointest. Liver Physiol. 32):
G548-G557,
1995[Abstract/Free Full Text].
20.
Lingwood, C. A.
Verotoxins and their glycolipid receptors.
Adv. Lipid Res.
25:
189-211,
1993[Medline].
21.
Martin, O. C.,
and
R. E. Pagano.
Internalization and sorting of a fluorescent analogue of glucosylceramide to the Golgi apparatus of human skin fibroblasts: utilization of endocytic and nonendocytic transport mechanisms.
J. Cell Biol.
125:
769-781,
1994[Abstract].
22.
Parton, R. G.
Ultrastructural localization of gangliosides; GM1 is concentrated in caveolae.
J. Histochem. Cytochem.
42:
155-166,
1994[Abstract/Free Full Text].
23.
Parton, R. G.
Caveolae and caveolins.
Curr. Opin. Cell Biol.
8:
542-548,
1996[Medline].
24.
Parton, R. G.,
C. D. Ockleford,
and
D. R. Critchley.
A study of the mechanism of internalisation of tetanus toxin by primary mouse spinal cord cultures.
J. Neurochem.
49:
1057-1068,
1987[Medline].
25.
Ramotar, K.,
B. Boyd,
G. Tyrrell,
J. Gariepy,
C. Lingwood,
and
J. Brunton.
Characterization of Shiga-like toxin I B subunit purified from overproducing clones of the SLT-I B cistron.
Biochem. J.
272:
805-811,
1990[Medline].
26.
Sandvig, K.,
and
J. E. Brown.
Ionic requirements for entry of Shiga toxin from Shigella dysenteriae 1 into cells.
Infect. Immun.
55:
298-303,
1987[Medline].
27.
Sandvig, K.,
O. Garred,
P. K. Holm,
and
B. van Deurs.
Endocytosis and intracellular transport of protein toxins.
Biochem. Soc. Trans.
21:
707-711,
1993[Medline].
28.
Sandvig, K.,
O. Garred,
K. Prydz,
J. V. Kozlov,
S. H. Hansen,
and
B. van Deurs.
Retrograde transport of endocytosed Shiga toxin to the endoplasmic reticulum.
Nature
358:
510-512,
1992[Medline].
29.
Sandvig, K.,
O. Garred,
and
B. van Deurs.
Thapsigargin-induced transport of cholera toxin to the endoplasmic reticulum.
Proc. Natl. Acad. Sci. USA
93:
12339-12343,
1996[Abstract/Free Full Text].
30.
Sandvig, K.,
K. Prydz,
S. H. Hansen,
and
B. van Deurs.
Ricin transport in brefeldin A-treated cells: correlation between Golgi structure and toxic effect.
J. Cell Biol.
115:
971-981,
1991[Abstract].
31.
Sandvig, K.,
M. Ryd,
O. Garred,
E. Schweda,
P. K. Holm,
and
B. van Deurs.
Retrograde transport from the Golgi complex to the ER of both Shiga toxin and nontoxic Shiga-B fragment is regulated by butyric acid and cAMP.
J. Cell Biol.
126:
53-64,
1994[Abstract].
32.
Saxena, S. K.,
A. D. O'Brien,
and
E. J. Ackerman.
Shiga toxin, Shiga-like toxin II variant, and ricin are all single-site RNA N-glycosidases of 28 S RNA when microinjected into Xenopus oocytes.
J. Biol. Chem.
264:
596-601,
1989[Abstract/Free Full Text].
33.
Simpson, J. C.,
C. Dascher,
L. M. Roberts,
J. M. Lord,
and
W. E. Balch.
Ricin cytotoxicity is sensitive to recycling between the endoplasmic reticulum and the Golgi complex.
J. Biol. Chem.
270:
20078-20083,
1995[Abstract/Free Full Text].
34.
Spangler, B. D.
Structure and function of cholera toxin and the related Escherichia coli heat-labile enterotoxin.
Microbiol. Rev.
56:
622-647,
1992[Abstract].
35.
van Weert, A. W.,
K. W. Dunn,
H. J. Gueze,
F. R. Maxfield,
and
W. Stoorvogel.
Transport from late endosomes to lysosomes, but not sorting of integral membrane proteins in endosomes, depends on the vacuolar proton pump.
J. Cell Biol.
130:
821-834,
1995[Abstract].
36.
Voorhees, P.,
E. Deignan,
E. van Donselaar,
J. Humphrey,
M. S. Marks,
P. J. Peters,
and
J. S. Bonifacino.
An acidic sequence within the cytoplasmic domain of furin functions as a determinant of trans-Golgi network localization and internalization from the cell surface.
EMBO J.
14:
4961-4975,
1995[Abstract].
AJP Cell Physiol 274(2):C319-C332
0363-6143/98 $5.00
Copyright © 1998 the American Physiological Society