Role of receptor-mediated endocytosis, endosomal
acidification and cathepsin D in cholera toxin cytotoxicity
Tatiana El Hage
1,2,
*, Cle
´
mence Merlen
1,2
*, Sylvie Fabrega
1,2
and Franc¸ois Authier
1,2
1 INSERM, U756, Cha
ˆ
tenay-Malabry, France
2 Universite
´
Paris-Sud, Faculte
´
de Pharmacie, Cha
ˆ
tenay-Malabry, France
Cholera toxin (CT) is the causative agent of the diarr-
heal disease cholera, and mediates its effects by
increasing cAMP levels [1]. The resulting increase in
intracellular cAMP causes net intestinal salt and
water secretion, resulting in massive secretory diarrhea
and changes in cell morphology, presumably due to
Keywords
acidification; cathepsin D; cholera toxin;
endosome; G protein

observed, coincident with massive internalization of both the 45 kDa and
47 kDa Gsa proteins. These events coincided with the endosomal recruit-
ment of ADP-ribosylation factor proteins, especially ADP-ribosylation fac-
tor-6, with a time course identical to that of toxin and the A subunit of the
stimulatory G protein (Gsa) translocation. After an initial lag phase of
30 min, these constituents were linked to NAD-dependent ADP-ribosyla-
tion of endogenous Gsa, with maximum accumulation observed at
30–60 min postinjection. Assessment of the subsequent postendosomal fate
of internalized Gsa revealed sustained endolysosomal transfer of the two
Gsa isoforms. Concomitantly, cholera toxin increased in vivo endosome
acidification rates driven by the ATP-dependent H
+
-ATPase pump and
in vitro vacuolar acidification in hepatoma HepG2 cells. The vacuolar H
+
-
ATPase inhibitor bafilomycin and the cathepsin D inhibitor pepstatin A
partially inhibited, both in vivo and in vitro, the cAMP response to cholera
toxin. This cathepsin D-dependent action of cholera toxin under the con-
trol of endosomal acidity was confirmed using cellular systems in which
modification of the expression levels of cathepsin D, either by transfection
of the cathepsin D gene or small interfering RNA, was followed by parallel
changes in the cytotoxic response to cholera toxin. Thus, in hepatic cells, a
unique endocytic pathway was revealed following cholera toxin administra-
tion, with regulation specificity most probably occurring at the locus of the
endosome and implicating endosomal proteases, such as cathepsin D, as
well as organelle acidification.
Abbreviations
ARF, ADP-ribosylation factor; CT, cholera toxin; CT-A, cholera toxin A subunit; CT-B, cholera toxin B subunit; ER, endoplasmic reticulum;
GSa, A subunit of the stimulatory G protein; LPS, postmitochondrial supernatant; si, small interfering.

maximum accumulation observed by 15–30 min [4,7].
Following ATP-dependent endosomal acidification,
internalized CT was rapidly proteolyzed within hepatic
endosomes by aspartic acid protease cathepsin D [7].
In vivo studies showed that the acidotropic agent
chloroquine, as well as the carboxylic ionophore
monensin, inhibited CT activation of adenylate cyclase
and increased the lag period for this process [5,6].
In vitro experiments revealed that hydrolysates of CT
generated by cathepsin D displayed ADP-ribosyltrans-
ferase activity towards exogenous Gs a [7]. However,
the mechanisms by which the endosome-activated
CT-A gains access to Gsa, which is mainly localized to the
inner face of the plasma membrane, remain undefined.
A second activating pathway has been proposed to
operate within the ER, which CT accesses by retrograde
vesicular traffic via the trans-Golgi network. In the
ER, the disulfide bond linking CT-A1 to CT-A2 ⁄
CT-B5 is reduced by protein disulfide isomerase, and
CT-A1 is then translocated to the cytosol in a process
involving ER-associated degradation. The cytosolic
pool of CT-A1 escapes ubiquitin-mediated protein deg-
radation, due to its very limited number of internal
lysine residues [8,9], and subsequently ADP-ribosylates
Gsa. However, mutagenesis studies have indicated that
although the ER retrieval signal of CT-A2 and the ER
localization of the toxin enhance the efficiency of CT
cytotoxicity, they are not absolutely required for toxin
action, suggesting the existence of alternative compart-
ment(s) for CT activation [10,11].

from control and CT-injected rats (Fig. 1). In agree-
ment with our previous work [7], a time-dependent
increase in CT-A and CT-B was observed in endoso-
mal fractions 10–20 min after native CT injection
(Fig. 1, upper left blot) or 20–90 min after CT-B injec-
tion (Fig. 1, upper right blot). In control rats,
immunoreactive Gsa was detected as a doublet of
47 kDa and 45 kDa (Fig. 1, lanes 1 of lower blots).
In vivo injection of native CT or CT-B effected a rapid
increase of both the 47 kDa and 45 kDa Gsa isoforms,
with maximal accumulation 20 min (native CT; 32%
increase) or 30 min (CT-B; 77% increase) postinjec-
tion. By 90 min postinjection, both Gsa isoforms had
returned to basal levels (Fig. 1, lane 6 of lower blots).
T. El Hage et al. Cytotoxic action of cholera toxin and cathepsin D
FEBS Journal 274 (2007) 2614–2629 ª 2007 The Authors Journal compilation ª 2007 FEBS 2615
Next, we used the in situ liver model system for
endosome–lysosome transfer analysis to determine the
endosomal fate of the internalized CT and Gsa
(Fig. 2). Transfer of CT and Gsa from the endosomal
compartment to the lysosomal compartment was
examined by Nycodenz density gradient analysis of the
postmitochondrial supernatant (LPS) fractions pre-
pared 20 min after CT administration (Fig. 2A). When
LPS fractions were incubated at 4 °C, most of the CT-
B and Gsa appeared in a single broad region with a
density of 1.077–1.119 gÆmL
)1
(Fig. 2A, left blots),
which mainly coincided with the Golgi marker galacto-

5–60 min postinjection (Fig. 3A, panel S).
Finally, we performed an in vivo CT substrate
labeling experiment using [
32
P]NAD and endocytic
vesicles that contained in vivo internalized native
Fig. 1. CT-mediated internalization of Gsa in the endosomal apparatus. Rat liver endosomal fractions were isolated at the indicated times
after the in vivo administration of native CT or CT-B, and evaluated by western blotting for their content of both CT subunits and Gsa. Fifty
micrograms of protein was applied to each lane. Molecular mass markers are indicated on the left of the upper panels. Arrows to the right
indicate the mobility of CT-A ( 28 kDa), CT-B ( 12 kDa) and Gsa ( 47 and 45 kDa). Lower panels: quantification of Gsa signals by scan-
ning densitometry, with results expressed as percentage of signal intensity in the endosomal fraction prepared from control (noninjected)
rats.
Cytotoxic action of cholera toxin and cathepsin D T. El Hage et al.
2616 FEBS Journal 274 (2007) 2614–2629 ª 2007 The Authors Journal compilation ª 2007 FEBS
CT (Fig. 3B). Radiolabeling of endosomal Gsa
was observed with endosomal fractions prepared 30
and 60 min postinjection of native CT. Thus, CT is
active in vivo towards endosomal Gsa following
a 30 min lag period, which probably corres-
ponds to the time required for its internalization
into endocytic structures and subsequent proteolytic
activation.
Effect of CT on ATP-dependent endosomal
acidification
It has been previously reported that 18 h after the
intraperitoneal injection of CT into rats, hepatic endo-
somes displayed increased rates of acidification and a
more acidic steady-state intravesicular pH [13]. There-
fore, we investigated whether CT altered endosomal
A

ing CT administration (closed squares). The initial rate
of ATP-dependent acidification of endosomes (which
was linearly related to incubation time for the first
5 min) increased two-fold in endosomes isolated from
CT-injected rats (closed squares), but this was not
observed for CT-B-injected (closed diamonds) or
diptheria toxin-injected rats (closed circles).
Bafilomycin A1 neutralizes endosomal acidification
by inhibiting the vacuolar ATPases responsible for
maintaining proton gradients [16]. It was therefore of
interest to determine whether bafilomycin A1 would
similarly affect endosomal acidification in control and
CT-treated cells (Fig. 4B). Incubation of HepG2 cells
for 30 min with bafilomycin A1 alone (0.2 lm) abol-
ished the granular fluorescence of DAMP almost com-
pletely (Fig. 4B, lower left panel). However, a residual
fluorescent staining reminiscent of vesicular acidifica-
tion was clearly observed in cells pretreated with
bafilomycin A1 and then incubated with CT for 2 h
(Fig. 4B, lower right panel). These data are consistent
with our finding that CT increased endosomal acidifi-
cation at the early stage of CT action.
Role of endosomal acidification and cathepsin D
in CT action
To assess whether the aspartic acid protease cathep-
sin D and endosomal acidity might be two major
requirements for CT cytotoxicity in hepatic cells, we
examined the in vivo and in vitro effects of agents that
inhibit aspartic acid protease activity and ⁄ or vesicle
acidification (Fig. 5). Animals were given an intraperi-

CT-B, and evaluated by western blotting for their content of ARF
and ARF-6 using their respective polyclonal and monoclonal anti-
bodies. Arrows to the right of each panel indicate the mobilities of
immunodetected ARF proteins ( 21 kDa). (B) Endosomal fractions
were isolated at the indicated times after the in vivo administration
of native CT, and immediately incubated with 0.54 l
M [
32
P]NAD at
30 °C in an ADP-ribosylation buffer; this was followed by
SDS ⁄ PAGE and autoradiography. Molecular mass markers are indi-
cated to the left of the panel. The arrow on the right indicates the
mobility of [
32
P]-labeled Gsa ( 45 kDa).
Cytotoxic action of cholera toxin and cathepsin D T. El Hage et al.
2618 FEBS Journal 274 (2007) 2614–2629 ª 2007 The Authors Journal compilation ª 2007 FEBS
Time of incubation (min)
control
control + ATP
cholera toxin
cholera toxin + ATP
diphtheria toxin
diphtheria toxin + ATP
A
30
15
200
400
Fluorescence Intensity (A.U.)

represents the mean ± SD of at least three
independent determinations. (B) HepG2
hepatoma cells were treated with CT
(10 lgÆmL
)1
) and incubated at 37 °C in the
absence (control) or presence of pepstatin A
(120 lgÆmL
)1
) or bafilomycin A1 (0.2 l M) for
the indicated times. Cellular cAMP content
was measured as described for (A), and
the data were expressed as pmolÆ(mG pro-
tein)
)1
. Results are the mean ± SD of three
separate experiments.
T. El Hage et al. Cytotoxic action of cholera toxin and cathepsin D
FEBS Journal 274 (2007) 2614–2629 ª 2007 The Authors Journal compilation ª 2007 FEBS 2619
As pepstatin A treatment produced, both in vivo
and in vitro, a sustained reduction in the cAMP
response to CT, we next evaluated the role of the
pepstatin A-sensitive enzyme cathepsin D in CT cyto-
toxicity by using cathepsin D-deficient 3Y1-Ad12 cells
transfected with the human cathepsin D gene [18].
Immunoblot analysis of equal amounts of protein
from 3Y1-Ad12 cell lysates confirmed the absence of
cathepsin D in nontransfected cells, and the presence
of both the 31 kDa mature cathepsin D and the
45 kDa procathepsin D in cathepsin D-overexpressing

of procathepsin D ( 45 kDa) and mature cathepsin D ( 31 kDa). (B) MCF-7 cells, whose cathepsin D expression was inhibited by siRNA
silencing for 48–72 h, were incubated with CT (1.3 l
M) for 2 h. Cellular cAMP content was measured and expressed as fold stimulation over
basal (unstimulated) activity [ 28 pmolÆ (mG protein)
)1
] (upper panel). Results are the mean ± SD of three separate experiments. Whole cell
lysates (60 lg of protein per lane) were evaluated by immunoblotting for their content of human cathepsin D (lower panel). Arrows on the
right indicate the mobility of procathepsin D ( 45 kDa) and mature cathepsin D ( 31 kDa).
Cytotoxic action of cholera toxin and cathepsin D T. El Hage et al.
2620 FEBS Journal 274 (2007) 2614–2629 ª 2007 The Authors Journal compilation ª 2007 FEBS
Assessment of the KDEL peptide integrity
of endosomal CT-A
The results presented thus far established that CT-A
represented a high-affinity substrate for endosomal
cathepsin D that assisted in the release of CT-A frag-
ment(s) that are active towards Gs a. However, the
endosomal degradative CT-A fragment(s) remained
undefined, and it was unknown whether the processed
form(s) of internalized CT-A had lost part or all of its
C-terminal ER-retention KDEL motif.
To investigate this, we characterized three polyclonal
antibodies to KDEL for their specificity towards CT-A
by western blot analysis of pure CT or CT-A
(Fig. 7A). Each antibody revealed a specificity for
CT-A, with the antibody to KAVKKDEL revealing
the highest affinity. Therefore, the antibody to
KAVKKDEL was used to assess the presence of the
KDEL peptide in the internalized CT-A. No percep-
tible KDEL immunoreactivity was detected in rat liver
endosomal fractions isolated 5–90 min post-CT injec-

(12-kDa)
0 5 15 30 60 90
CT
B
α-CT
0 5 15 30 60 90
CT
α-KAVKKDELα-CT
CT-A
(28-kDa)
CT-B
(12-kDa)
–
45
4
pH–
–3030
60 Time of incubation (min)
–
C
Fig. 7. Metabolic fate of the KDEL peptide during endosomal proteolysis of internalized CT. (A) Polyclonal sera against CT or KDEL peptides
were assessed by western blotting for their ability to bind specifically to CT-A. Each lane contained 1 lg of CT-A or 5 lg of CT. Arrows to
the left indicate the mobilities of CT-A ( 28 kDa) and CT-B ( 12 kDa). Each antibody to KDEL showed specificity for CT-A. Polyclonal anti-
serum to CT bound to both subunits. (B) Rat liver endosomal fractions were isolated at the indicated times after the in vivo administration of
CT, and evaluated by western blotting for their immunoreactivity using polyclonal antiserum to synthetic peptide KAVKKDEL (a-KAVKKDEL)
or polyclonal IgG against CT (a-CT) (incubation with the same membrane). Fifty micrograms of protein was applied to each lane. Arrows to
the left indicate the mobilities of CT-A ( 28 kDa) and CT-B ( 12 kDa). (C) Endosomal fractions were incubated with 10 lg of native CT at
37 °C for the indicated times in 30 m
M citrate ⁄ phosphate buffer at the indicated pH. The incubation mixtures were then analyzed by western
blotting using polyclonal antibody to CT (left panel) or polyclonal antibody to KAVKKDEL (right panel). The mobilities of each intact CT subunit

systems, we have obtained evidence that the cAMP
response in CT-treated cells was, at least in part, rela-
ted to the proteolytic activity and expression level of
the aspartic acid protease cathepsin D (Fig. 8). How-
ever, internalized CT that has been localized within the
ER in murine hepatocyte BNL CL.2 cells [19] may
also follow another activating pathway operating at a
late stage of endocytosis and requiring retrograde
transport.
Although G proteins are widely accepted as media-
tors of signal transduction by cell surface receptors,
several lines of evidence now indicate that trimeric
G proteins are located in the endosomal compartment
of various cells and are involved in vesicular transport
events through the endocytic pathway [20]. Consistent
with previous studies [21,22], similar amounts of two
Fig. 8. Endosomal regulation of CT activation and action in rat liver.
Cytotoxic action of cholera toxin and cathepsin D T. El Hage et al.
2622 FEBS Journal 274 (2007) 2614–2629 ª 2007 The Authors Journal compilation ª 2007 FEBS
forms of Gsa, with apparent molecular masses of
47 kDa (large form, Gsa-L) and 45 kDa (small form,
Gsa-S), have been identified in our endosomal frac-
tions isolated from noninjected rats. These isoforms
are produced by alternative splicing of a single precur-
sor mRNA [23].
The translocation of G proteins from the plasma
membrane to the endosomal apparatus has been demon-
strated biochemically and morphologically in various
cells stimulated by agonists such as glucagon [21], iso-
proterenol [24], carbachol [25], thyrotropin-releasing

peptide or CT-A fragment may partition spontane-
ously into the hydrophobic core of the endosomal
membrane. Fluorescence resonance energy transfer,
used to monitor pH-dependent structural changes in
CT-B, has revealed that the low endosomal pH is cap-
able of inducing structural changes in CT, which, in
turn, exerts its effect on the structure of the membrane
to which CT-B is bound [33]. The role of endosomal
acidity in facilitating CT-A translocation across the
endosomal membrane has also been demonstrated
using hepatic endosomes isolated after injection of
native CT, and then examined for their ability to bind
antibodies to CT-A and to stimulate exogenous plasma
membrane-associated adenylate cyclase [6]. Time- and
acid-dependent exteriorization of CT-A was observed
with no translocation of CT-B [6]. These findings
would be consistent with a model in which CT mark-
edly increases endosomal acidification rates (this study)
[13], to allow maximal insertion of activated CT-A into
the endosomal membrane, leading to efficient ADP-
ribosylation of cointernalized Gsa.
The enzymatic activity of CT-A1 is allosterically sti-
mulated by ARFs, which are host-cell small GTP-
binding proteins active in the GTP-bound form [34].
On the basis of the reconstitution of a signal transduc-
tion pathway in a bacterial two-hybrid system, a direct
interaction between human ARF-6 (belonging to the
class III ARFs) and CT-A1 was demonstrated [34].
Recently, the cocrystallization of CT and ARF-6 has
defined the structural basis for activation of CT by

125
I]CT uptake into rat liver
have previously shown that some radioactivity (30 min
to 2 h postinjection) is intrinsic to acid-phosphatase-
containing structures, presumably lysosomes [4]. Using
the in situ rat liver model system for endosome–
lysosome fusion, we have confirmed a low lysosomal
T. El Hage et al. Cytotoxic action of cholera toxin and cathepsin D
FEBS Journal 274 (2007) 2614–2629 ª 2007 The Authors Journal compilation ª 2007 FEBS 2623
transfer of internalized native CT-B, which was accom-
panied by a sustained transfer of Gsa, the CT sub-
strate. The endolysosomal transfer of CT-B was also
accompanied by a net decrease in its immunoreactivity
throughout the gradient, suggesting that CT-B is pro-
teolyzed in the endosomal apparatus (this study) [7], as
well as within lysosomal vesicles. Despite the fact that
the catalytic CT-A was below the limits of detection in
our fusion system, the massive cotransfer of Gsa to
lysosomes observed in response to CT injection sug-
gests that extended ADP-ribosylation of Gsa may also
occur in vivo at the locus of the lysosomal apparatus.
Weak bases and proton ionophores have been used
in vivo and in vitro to study the role of organelle
acidification in the cytotoxic action of CT [5,6]. Using
isolated rat hepatocytes, it has been shown that
chloroquine inhibits the ability of CT to activate
adenylate cyclase, and that a similar effect occurs with
monensin [5]. In addition, both drugs comparably
inhibited generation of the CT-A1 peptide. In rat liver,
chloroquine accumulates in hepatic endosomes, leading

Our in vivo and in vitro studies with hepatocytes are in
marked contrast to in vitro studies using other cellular
models, where acidotropic drugs and V-ATPase inhibi-
tors had little to no effect on the cytotoxic action of
CT [42,43]. This may be a reflection of the differences
between hepatocytes and other cell types.
Previous studies using rat liver have shown that CT
markedly alters several aspects of fluid-phase endo-
cytosis: it increases rates of endosome acidification
without altering ion conductances, and leads to a more
acidic steady-state intraendosomal pH, persistence of
Na
+
⁄ H
+
exchange in late endosomes, and changes in
endosome trafficking [13,30]. In contrast, CT had no
significant effect on lysosome acidification rates and
steady-state internal pH, indicating that CT predomin-
antly altered the earlier steps of endocytosis [44]. How-
ever, the effects of CT on endosome acidification were
demonstrated at a late exposure time, with animals
receiving CT intraperitoneally 16 h prior to endosome
preparation [13]. Our estimate of the rate of endosom-
al acidification, which increased two-fold after CT
treatment, compared favorably with these previous
reports [13,30]. Moreover, we provide three supple-
mental observations: (a) endosomal acidification rates
were significantly increased 2 h post-CT treatment,
which coincided with the presence of internalized CT

number of H
+
-ATPase pumps per endosome and ⁄ or
redistribution of vacuolar H
+
-pumps. Whatever the
precise signal transduction mechanism responsible for
CT increasing endosome H
+
transport, our data sug-
gest that the more acidic pH of endocytic vesicles at
Cytotoxic action of cholera toxin and cathepsin D T. El Hage et al.
2624 FEBS Journal 274 (2007) 2614–2629 ª 2007 The Authors Journal compilation ª 2007 FEBS
an early stage may be part of the toxic action of CT,
facilitating the proteolytic activation of CT-A by cath-
epsin D, and its subsequent translocation across the
endosomal membrane, both of which require an acidic
pH [5,6].
Although suggesting that it is the CT–CT receptor
complex that is internalized to the endolysosomal
apparatus of hepatocytes, our studies described here
provide no direct information on the nature and fate of
the CT receptor. Using an in vivo biochemical approach
similar to that used to study the hepatic fate of CT (this
study) [7], studies are currently underway to elucidate
whether the CT receptor is rapidly and specifically
internalized with its ligand to low-density endosomes.
Experimental procedures
Peptides, antibodies, protein determination,
enzyme assays and materials

kit were obtained from Amersham. Nycodenz was obtained
from Nycomed Pharma. DAMP was obtained from
Molecular Probes. All other chemicals were obtained from
commercial sources and were of reagent grade.
Animals and injections
In vivo procedures were approved by the institutional com-
mittee for use and care of experimental animals. Male Spra-
gue-Dawley rats, body weight 180–200 g, were obtained
from Charles River France (St Aubin Les Elbeufs, France)
and were fasted for 18 h prior to being killed. Native CT,
CT-B or diphtheria toxin (50 lg per 100 g body weight) in
0.4 mL of 0.15 m NaCl was injected within 5 s into the
penile vein under light anesthesia with ether.
Isolation of subcellular fractions from rat liver
Subcellular fractionation was performed using established
procedures [7]. Following injection of toxins, rats were killed,
and the livers were rapidly removed and minced in isotonic
ice-cold homogenization buffer as previously described [7].
Rat liver cytosolic fraction was isolated by differential
centrifugation as previously described [48–50]. The plasma
membrane fraction was isolated from the nuclear fraction
as described by Hubbard et al. [51]. The endosomal fraction
was isolated by discontinuous sucrose gradient centrifuga-
tion, and collected at the 0.25–1.0 m sucrose interface
[7,17,48–50]. The soluble endosomal extract was isolated
from the endosomal fraction by freeze–thawing in 5 mm
sodium phosphate (pH 7.4), disrupted in the same hypo-
tonic medium using a small Dounce homogenizer (15
strokes with a Type A pestle), and centrifuged at 150 000 g
for 60 min in a Beckman 70.1 Ti rotor as previously des-

against human ARF-1 (diluted 1 : 400) and Gsa (diluted
1 : 10 000) and mouse monoclonal IgG against human
ARF-6 (diluted 1 : 100)] in the above buffer for 16 h at
4 °C. The blots were then washed three times with 0.5%
skimmed milk in 10 mm Tris ⁄ HCl (pH 7.5), 300 mm NaCl
and 0.05% Tween-20 over a period of 1 h at room tem-
perature. The bound immunoglobulin was detected using
horseradish peroxidase-conjugated goat anti-(rabbit) IgG.
CT-catalyzed ADP-ribosylation
Hepatic endosomal fractions were prepared from CT-injected
rats. The endosomal fractions ( 50 lg) were then suspended
in an ADP-ribosylation buffer containing 0.54 lm
[
32
P]NAD, 50 mm sodium phosphate buffer (pH 7.2),
0.5 mm GTP, 1 mm ATP, 5 mm MgCl
2
, and 10 mm thymi-
dine, and incubated at 30 °C for 45 min. The reaction was
stopped by the addition of Laemmli sample buffer [54], and
this was followed by SDS ⁄ PAGE and autoradiography.
Cell-free assay for ATP-dependent endosomal
acidification
ATP-dependent acidification of isolated endosomes was
assayed using acridine orange, a membrane-permeable lipo-
philic weak base that accumulates in acidic organelles [15].
Hepatic endosomes isolated after injection of native cholera
or diphtheria toxins (120 lg) were incubated in 0.15 m
KCl, 5 mm MgCl
2

)1
) or bafilomycin A1
(0.2 lm). Control cells received 2% dimethylsulfoxide only.
At the end of the incubation, the medium was removed,
cells were lysed with 1 m perchloric acid, and cAMP was
measured as above [13].
Cell culture
Human hepatoma (HepG2) cells were grown in DMEM
supplemented with 10% (v ⁄ v) fetal bovine serum and 1%
penicillin ⁄ streptomycin in an atmosphere of 95% air ⁄ 5%
CO
2
[55]. The human breast carcinoma cell line MCF-7
was grown in DMEM supplemented with 10% (v ⁄ v) fetal
bovine serum in an atmosphere of 90% air ⁄ 10% CO
2
[56].
Cathepsin D-transfected 3Y1-Ad12 carcinoma cells were
grown in RPMI medium supplemented with 5% fetal
bovine serum, 400 lgÆmL
)1
G418 and 50 lgÆmL
)1
gentami-
cin in an atmosphere of 95% air ⁄ 5% CO
2
[18].
Transfection and RNA interference
Cathepsin D siRNA and control siRNA were synthesized
by Eurogentec (Seraing, Belgium). The siRNA sequence for

lized with 0.1% Triton X-100 in NaCl ⁄ P
i
for 4 min and
0.5% saponine in NaCl ⁄ P
i
for 10 min. Permeabilized cells
were blocked for 20 min with 10% horse serum in NaCl ⁄ P
i
,
and then incubated for 60 min with a rabbit polyclonal
anti-dinitrophenyl fluorescein conjugate (1 : 100 dilution).
Cytotoxic action of cholera toxin and cathepsin D T. El Hage et al.
2626 FEBS Journal 274 (2007) 2614–2629 ª 2007 The Authors Journal compilation ª 2007 FEBS
Laser-scanning confocal microscopy was performed using a
Zeiss LSM 510 confocal (Axiovert 100 m) inverted micro-
scope equiped with a Zeiss X63 ⁄ 1.4 NA oil immersion
objective lens (plan-Apochromat).
Acknowledgements
We thank Pamela H. Cameron (McGill University,
Montreal, Quebec, Canada) for reviewing the manu-
script and assistance in these studies. We thank
Dr F. Nato (Institut Pasteur, Paris, France) for the
kind gift of monoclonal antibodies to CT, Dr V. Nico-
las (IFR 75 INSERM, Faculte
´
de Pharmacie, Chaˆ te-
nay-Malabry, France) for assistance with confocal
microscopy, Dr C. Rouyer-Fessard (INSERM U773,
Faculte
´

requires toxin internalization and processing in endo-
somes. J Biol Chem 266, 12858–12865.
7 Merlen C, Fayol-Messaoudi D, Fabrega S, El Hage T,
Servin A & Authier F (2005) Proteolytic activation of
internalized cholera toxin within hepatic endosomes by
cathepsin D. FEBS J 272, 4385–4397.
8 Hazes B & Read RJ (1997) Accumulating evidence sug-
gests that several AB-toxins subvert the endoplasmic
reticulum-associated protein degradation pathway to
enter target cells. Biochemistry 36, 11051–11054.
9 Rodighiero C, Tsai B, Rapoport TA & Lencer WI
(2002) Role of ubiquitination in retro-translocation of
cholera toxin and escape of cytosolic degradation.
EMBO Rep 3, 1222–1227.
10 Cieplak W, Mead DJ, Messer RJ & Grant CC (1995)
Site-directed mutagenic alteration of potential active-site
residues of the A subunit of Escherichia coli heat-labile
enterotoxin. Evidence for a catalytic role for glutamic
acid 112. J Biol Chem 270, 30545–30550.
11 Lencer WI, Constable C, Moe S, Jobling MG, Webb
HM, Ruston S, Madara JL, Hirst TR & Holmes RK
(1995) Targeting of cholera toxin and Escherichia coli
heat labile toxin in polarized epithelia: role of COOH-
terminal KDEL. J Cell Biol 131, 951–962.
12 Teter K, Jobling MG, Sentz D & Holmes RK (2006)
The cholera toxin A1 (3) subdomain is essential for
interaction with ADP-ribosylation factor 6 and full
toxic activity but is not required for translocation from
the endoplasmic reticulum to the cytosol. Infect Immun
74, 2259–2267.

Authier F (2006) Glucagon-mediated internalization of
serine-phosphorylated glucagon receptor and Gsa in rat
liver. FEBS Lett 580, 5697–5704.
22 Van Dyke RW (2004) Heterotrimeric G protein sub-
units are located on rat liver endosomes.
BMC Physiol
4, 1–16.
23 Yagami T, Kirita S, Matsushita A, Kawasaki K &
Mizushima Y (1994) Alterations in the stimulatory G
T. El Hage et al. Cytotoxic action of cholera toxin and cathepsin D
FEBS Journal 274 (2007) 2614–2629 ª 2007 The Authors Journal compilation ª 2007 FEBS 2627
protein of the rat liver after partial hepatectomy. Bio-
chim Biophys Acta 1222, 81–87.
24 Wedegaertner PB, Bourne HR & von Zastrow M (1996)
Activation-induced subcellular redistribution of Gs
alpha. Mol Biol Cell 7, 1225–1233.
25 Svoboda P & Milligan G (1994) Agonist-induced trans-
fer of the A rtbtmiss of the guanine-nucleotide-binding
regulatory proteins Gq and G11 and of muscarinic m1
acetylcholine receptors from plasma membranes to a
light-vesicular membrane fraction. Eur J Biochem 224,
455–462.
26 Svoboda P, Kim GD, Grassie MA, Eidne KA &
Milligan G (1996) Thyrotropin-releasing hormone-
induced subcellular redistribution and down-regulation
of G11a: analysis of agonist regulation of coex-
pressed G11a species variants. Mol Pharmacol 49,
646–655.
27 Arthur JM, Collinsworth GP, Gettys TW & Raymond
JR (1999) Agonist-induced translocation of Gq ⁄ 11a

34 Jobling MG & Holmes RK (2000) Identification of
motifs in cholera toxin A1 polypeptide that are required
for its interaction with human ADP-ribosylation factor
6 in a bacterial two-hybrid system. Proc Natl Acad Sci
USA 97, 14662–14667.
35 O’Neal CJ, Jobling MG, Holmes RK & Hol WGJ
(2005) Structural basis for the activation of cholera
toxin by human AR6-GTP. Science 309, 1093–1096.
36 Massol RH, Larsen JE, Fujinaga Y, Lencer WI &
Kirchhausen T (2004) Cholera toxin toxicity does not
require functionnal Arf6- and dynamin-dependent endo-
cytic pathways. Mol Biol Cell 15, 3631–3641.
37 Cavenagh MM, Whitney JA, Caroll K, Zhang C,
Boman AC, Rosenwald AG, Melhnan I & Kahn RA
(1996) Intracellular distribution of Arf proteins in mam-
malian cells. Arf6 is uniquely localized to the plasma
membrane. J Biol Chem 271, 21767–21774.
38 Yang CZ, Heimberg H, D’Souza-Schorey C, Mueckler
MM & Stahl PD (1998) Subcellular distribution and
differential expression of endogenous ADP-ribosylation
factor 6 in mammalian cells. J Biol Chem 273,
4006–4011.
39 D’Souza-Schorey C, van Donselaar E, Hsu VW, Yang
C, Stahl PD & Peters PJ (1998) ARF6 targets recycling
vesicles to the plasma membrane: insights from an ultra-
structural investigation. J Cell Biol 140, 603–616.
40 Maranda B, Brown D, Bourgoin S, Casanova JE, Vinay
P, Ausiello DA & Marshansky V (2001) Intra-endo-
somal pH-sensitive recruitment of the Arf-nucleotide
exchange factor ARNO and Arf6 from cytoplasm to

JJM (1994) Endosomal proteolysis of insulin by an
Cytotoxic action of cholera toxin and cathepsin D T. El Hage et al.
2628 FEBS Journal 274 (2007) 2614–2629 ª 2007 The Authors Journal compilation ª 2007 FEBS
acidic thiol metalloprotease unrelated to insulin-degrad-
ing enzyme. J Biol Chem 269, 3010–3016.
49 Authier F, Mort JS, Bell AW, Posner BI & Bergeron
JJM (1995) Proteolysis of glucagon within hepatic endo-
somes by membrane-associated cathepsins B and D.
J Biol Chem 270, 15798–15807.
50 Authier F, Me
´
tioui M, Bell AW & Mort JS (1999)
Negative regulation of epidermal growth factor signal-
ing by selective proteolytic mechanisms in the endo-
some mediated by cathepsin B. J Biol Chem 274,
33723–33731.
51 Hubbard AL, Wall DA & Ma A (1983) Isolation of rat
hepatocyte plasma membranes. 1. Presence of three
major domains. J Cell Biol 96, 217–229.
52 Chauvet G, Tahiri K, Authier F & Desbuquois B
(1998) Endosome–lysosome transfer of insulin and
glucagon in a liver cell-free system. Eur J Biochem 254,
527–537.
53 Authier F & Chauvet G (1999) In vitro endosome–
lysosome transfer of dephosphorylated EGF receptor
and Shc in rat liver. FEBS Lett 461, 25–31.
54 Laemmli UK (1970) Cleavage of structural proteins
during the assembly of the head of bacteriophage T4.
Nature 227, 680–685.
55 Authier F, Cameron PH & Taupin V (1996)