Proteolysis of Pseudomonas exotoxin A within hepatic
endosomes by cathepsins B and D produces fragments
displaying in vitro ADP-ribosylating and apoptotic effects
Tatiana El Hage
1,2
,Se
´
verine Lorin
3
, Paulette Decottignies
4,5
, Mojgan Djavaheri-Mergny
6
and
Franc¸ois Authier
1,2
1 INSERM, Cha
ˆ
tenay-Malabry, France
2 Universite
´
Paris-Sud, Faculte
´
de Pharmacie, Cha
ˆ
tenay-Malabry, France
3 JE 2493, Universite
´
Paris-Sud, Faculte
´
de Pharmacie, Cha

To assess Pseudomonas exotoxin A (ETA) compartmentalization, process-
ing and cytotoxicity in vivo, we have studied the fate of internalized ETA
with the use of the in vivo rodent liver model following toxin administra-
tion, cell-free hepatic endosomes, and pure in vitro protease assays. ETA
taken up into rat liver in vivo was rapidly associated with plasma mem-
branes (5–30 min), internalized within endosomes (15–60 min), and later
translocated into the cytosolic compartment (30–90 min). Coincident with
endocytosis of intact ETA, in vivo association of the catalytic ETA-A sub-
unit and low molecular mass ETA-A fragments was observed in the
endosomal apparatus. After an in vitro proteolytic assay with an endoso-
mal lysate and pure proteases, the ETA-degrading activity was attributed
to the luminal species of endosomal acidic cathepsins B and D, with the
major cleavages generated in vitro occurring mainly within domain III of
ETA-A. Cell-free endosomes preloaded in vivo with ETA intraluminally
processed and extraluminally released intact ETA and ETA-A in vitro in a
pH-dependent and ATP-dependent manner. Rat hepatic cells underwent
in vivo intrinsic apoptosis at a late stage of ETA infection, as assessed by
the mitochondrial release of cytochrome c, caspase-9 and caspase-3 activa-
tion, and DNA fragmentation. In an in vitro assay, intact ETA induced
ADP-ribosylation of EF-2 and mitochondrial release of cytochrome c, with
the former effect being efficiently increased by a cathepsin B ⁄ cathepsin D
pretreatment. The data show a novel processing pathway for internalized
ETA, involving cathepsins B and D, resulting in the production of
ETA fragments that may participate in cytotoxicity and mitochondrial
dysfunction.
Abbreviations
DT, diphtheria toxin; EEA1, early endosome antigen-1; EF-2, elongation factor-2; ER, endoplasmic reticulum; ETA, exotoxin A;
HA, hexa-
D-arginine; LRP1, low-density lipoprotein receptor-related protein 1; PA, pepstatin-A; SD, standard deviation;
a

This is initiated by cell surface binding of ETA to
the a
2
MG ⁄ LRP1 receptor [4], which is followed by
internalization of the toxin–receptor complex to the
endosomal apparatus by clathrin-dependent and clath-
rin-independent mechanisms [7]. Two subcellular com-
partments have been proposed as being physiologically
relevant to the mechanism of translocation of internal-
ized ETA into the cytosol. The first translocation path-
way has been proposed to operate at an early stage of
endocytosis from endocytic vesicles [8,9]. Thus, signifi-
cant translocation of ETA across the endosomal mem-
brane of mouse lymphocytes was demonstrated, and
required exposure of ETA to low endosomal pH and
ATP hydrolysis [10]. Other studies have proposed that
internalized ETA can be retrogradely transported to
the endoplasmic reticulum (ER) for retrotranslocation
to the cytosol through the Sec61 complex [11]. The ER
trafficking pathway of ETA might have multiple routes
[7], one being the previously characterized KDEL
pathway involving the REDLK C-terminal sequence
of the toxin [12].
Whatever the pathway enabling cytosolic delivery of
ETA, activating processes have been proposed to occur
at various stages of ETA trafficking. These activating
steps include furin-mediated cleavage at the Arg279-
Glu280 peptide bond [13], reduction of the disulfide
bond linking Cys265 and Cys287 [14], and removal of
the C-terminal Lys [15]. Thus, for full ADP-ribosyla-

(slow association). Our results assign an important role
to endosomal acidic cathepsins B and D in generating
ETA fragments displaying high in vitro ADP-ribosyl-
transferase activity towards cytosolic EF-2. We report
on the in vivo association of ETA and ETA-A with
cytosolic fractions, and the in vitro ATP-dependent
and pH-dependent translocation of ETA and ETA-A
from cell-free endosomes into the external milieu.
Finally, the mitochondrial release of cytochrome c,
activation of caspase-9 and caspase-3 and DNA frag-
mentation were detected in cytosolic fractions isolated
2 h after ETA treatment, relating for the first time
activation of the intrinsic apoptotic pathway with ETA
cytotoxicity in a physiological state.
Results
In vivo endocytosis and metabolic fate of ETA in
rat liver
The kinetics of in vivo uptake of ETA at the hepatic
cell surface (plasma membranes) (Fig. 1A) and intra-
cellularly (endosomes) (Fig. 1B) were assessed first.
Rats were given an intravenous injection of native
Proteolysis of ETA in rat hepatic endosomes T. El Hage et al.
3736 FEBS Journal 277 (2010) 3735–3749 ª 2010 The Authors Journal compilation ª 2010 FEBS
ETA (15 lg per 100 g body weight) and killed 5–90 min
postinjection. Following preparation of hepatic subcel-
lular fractions, the amount of internalized ETA was
determined by SDS ⁄ PAGE followed by western blot
analyses with antibody directed against ETA-A. It was
assumed that the in vivo generation of free ETA-A was
attributable to both reductive and proteolytic cleavages

tail epitope was found in the endosomal fraction iso-
lated from control rats. The extensive fragmentation of
LRP1 within hepatic endosomes may explain, in part,
the failure to detect intact LRP1 by us (this study) and
others [20]. In vivo injection of native ETA effected a
rapid increase in endosomal truncated LRP1, with
maximal accumulation at 5–15 min postinjection. By
60 min postinjection, the 80 kDa LRP1 species had
returned to basal levels (Fig. 2A, upper blot). How-
ever, the level of the endosomal marker early endo-
some antigen-1 (EEA1) was not modified after ETA
treatment (Fig. 2A, lower blot).
LRP1 enables endocytosis of ETA and various other
ligands among such as a
2
MG [21]. To examine the
effect of a
2
MG on the uptake of ETA into hepatic
endosomes, a
2
MG (15 lg per 100 g body weight) was
coinjected with ETA into rats (Fig. 2B). Endosomal
association of intact ETA and ETA-A was reduced by
a
2
MG coinjection.
We have previously reported that antibodies reacting
with the ER-retention KDEL motif are useful in
assessing the integrity of the C-terminal region of chol-

– 25
– 50
– 37
– 25
kDa
– 15
kDa
– 15
(min, postinjection)
AB
Fig. 1. Kinetics of appearance of ETA in hepatic plasma membranes and endosomes after toxin administration. Rat hepatic plasma mem-
brane (A) and endosomal fractions (B) were isolated at the indicated times after the in vivo administration of native ETA, and evaluated for
their content of internalized toxin by nonreducing (upper blots) and reducing SDS ⁄ PAGE (lower blots) followed by western blot analysis with
the polyclonal antibody against ETA. Each lane contained 10 lg (plasma membranes) or 30 l g (endosomes) of protein. The arrows to the left
of each panel indicate the mobilities of intact ETA ( 66 kDa), ETA-A ( 37 kDa), and unknown degradation fragments. Molecular mass
markers are indicated to the right of the reducing blots. The antibody against ETA also binds to undefined plasma membrane proteins dis-
tinct from ETA under nonreducing conditions [upper blot in (A)] in both control and toxin-injected rats, one of which had a molecular mass
identical to that of ETA-A.
T. El Hage et al. Proteolysis of ETA in rat hepatic endosomes
FEBS Journal 277 (2010) 3735–3749 ª 2010 The Authors Journal compilation ª 2010 FEBS 3737
against KDEL bind to the ETA C-terminal sequence
REDLK (which resembles the ER motif KDEL), we
first characterized antibodies against KDEL for their
binding to native ETA and ETA-A by western blot
analysis (Fig. 2C, left and middle blots). One antibody,
anti-KX
5
KDEL, bound to ETA-A but not to native
ETA (Fig. 2C, left and middle blots), whereas the
others, anti-KSEKKDEL and anti-KAVKKDEL, did

*
100
Arbitrary units
_
5 15 30 60 90 (min, postinjection)
ETA
ETA/a
2
MG
15 30 5 15 30 (min, postinjection)
ETA-A
(37 kDa)
ETA
(66 kDa)
5
– 100
– 75
– 50
– 37
– 25
– 15
kDa
α-EEA1
EEA1
(180 kDa)
_
5 15 30 60 90 (min, postinjection)
ETA
5153060
ETA

by western blot analysis. Each lane contained 30 lg(a-LRP1 blot)
or 50 lg(a-EEA1 blot) of endosomal protein. The LRP1 bands were
quantified by scanning densitometry, and the signal intensities for
the ETA-treated rats were expressed as a percentage (mean ± SD)
of the signal intensity for the control rats (lane )). *P < 0.05 for the
differences between ETA ⁄ 5 min or ETA ⁄ 15 min and control rats
()). The arrows to the right indicate the mobilities of membrane-
bound LRP1 fragment ( 80 kDa) or EEA1 ( 180 kDa). Uncleaved
LRP1 ( 600 kDa) was not observed in endosomal fractions from
control and toxin-injected rats. (B) Effect of a
2
MG treatment on the
internalization of ETA. Rat hepatic endosomal fractions were iso-
lated at the indicated times after the in vivo coadministration of
ETA and a
2
MG (15 lg per 100 g body weight), and evaluated for
their content of internalized toxin by reducing SDS ⁄ PAGE followed
by western blot analysis with the polyclonal antibody against ETA.
Each lane contained 50 lg of endosomal protein. The arrows to the
left indicate the mobilities of intact ETA ( 66 kDa), ETA-A
( 37 kDa), and unknown degradation fragments. Molecular mass
markers are indicated to the right. (C) Assessment of immunoreac-
tivity of antibody against KDEL for native and internalized ETA. ETA
was either untreated (left blot, lane ) ) or digested in vitro with
100 UÆmL
)1
Æmg
)1
furin and 10 mM dithiothreitol (middle blot,

tion for intact ETA, whereas no degradation was
observed for ETA-A (Fig. 3A).
We next examined the effects of various protease
inhibitors on the proteolysis of endosomal ETA and
ETA-A, using cell-free endosomes preloaded with ETA
toxin in vivo and incubated in vitro at pH 7 in the pres-
ence of ATP (Fig. 3B). Western blot analysis with the
antibody against ETA revealed that the endosomal
ETA-degrading activity was partially inhibited by the
aspartic acid protease inhibitor pepstatin-A (PA), the
cysteine protease inhibitor E64, and the metallopro-
tease inhibitor EDTA.
The inhibition of ETA-degrading activity by PA and
E64, its low pH optimum and its presence in the
endosomal lumen as a soluble form (results not shown)
suggested cathepsins B and D as likely candidates for
this activity. We therefore examined the hydrolysis of
ETA by pure cathepsins B and D at pH 4–7 (Fig. 3C).
pH 7 + ATP
_
PA
HA
EDTA
PMSF
E64
_
06060606060
60 (min)
(Inhibitor)
(

100
Arbitrary units
_
+
_
+
_
+ (ATP)
A
B
C
Fig. 3. Assessment of ETA-degrading activity associated with
hepatic endosomes. (A) Rat hepatic endosomal fractions were iso-
lated 30 min after ETA administration (15 lg per 100 g body
weight) and incubated for the indicated times at 37 °C in isotonic
buffer containing 0.15
M KCl, 25 mM Hepes (pH 7), 5 mM MgCl
2
,
and 6 m
M CaCl
2
, in the presence or absence of 10 mM ATP. The
integrity of ETA was then evaluated by reducing SDS ⁄ PAGE fol-
lowed by western blotting with the polyclonal antibody against
ETA. Each lane contained 50 lg of endosomal protein. The arrows
to the right indicate the mobilities of intact ETA ( 66 kDa), ETA-A
( 37 kDa), and unknown degradation fragments. Molecular mass
markers are indicated to the left. ETA and ETA-A signals were
quantified by scanning densitometry, and the signal intensities after

and 10 mM dithiothreitol for the indicated times. The integrity
of ETA was then evaluated by reducing SDS ⁄ PAGE followed
by western blotting with the polyclonal antibody against ETA.
The arrows to the left and right indicate the mobilities of intact
ETA ( 66 kDa), ETA-A ( 37 kDa), and unknown degradation
fragments.
T. El Hage et al. Proteolysis of ETA in rat hepatic endosomes
FEBS Journal 277 (2010) 3735–3749 ª 2010 The Authors Journal compilation ª 2010 FEBS 3739
Western blot analysis with an antibody against ETA
showed that cathepsins B and D degraded ETA in a
pH-dependent manner, with maximal degradation
being observed at pH 4. The ETA fragments generated
by the pure cathepsins (especially cathepsin B at pH 4)
had molecular masses very similar to those seen with
the endosomal fractions.
We then assessed the major proteolytic cleavages
induced by cathepsin B and ⁄ or D within the ETA
sequence at various pH values (Fig. 4A,B). The prote-
olysis of ETA at pH 4 or 6 by cathepsin B and ⁄ or D
was analyzed by reducing SDS⁄ PAGE (Fig. 4A), and
the cleavage sites in the major metabolites were deter-
mined by N-terminal sequence analysis (Fig. 4B).
Edman degradation of intermediates 4a, 4b, 4d, 6a
and 6c revealed the N-terminal sequence of ETA
(AEEAFDL), suggesting that the cleavage sites are
located within the C-terminal region of the toxin.
N-terminal sequence analysis of ETA fragments 6b
and 6d, generated at pH 6, revealed cleavages
between Thr396 and Cys397 (as demonstrated by the
CPVAAGECA sequence). For peptide 4c, generated

– 75
kDa
ETA
(66 kDa)
+ + + + Cathepsin B
+ + + Cathepsin D
4 6 4 6 4 pH of medium
ETA
1
– 50
– 37
– 25
– 15
4b
4c
6c
4a
6b
6a
6d
4d
0.25 3 0.25 3 0.25
Incubation time (h)
AEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDN
ALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGN
QLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISHAGVSVVMAQTQPRREKR
WSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRL
HFPEGGSLAALTAHQACHLPLETFTRHRQPR
279
1

sponding change in the toxin cytotoxicity towards
cytosolic EF-2 would be observed (Fig. 6A). ETA was
first partially processed by a mixture of cathepsins B
and D at pH 4 or 6, and then incubated at neutral pH
with cytosolic EF-2 in the presence of [
32
P]NAD. A
low level of ADP-ribosylation of EF-2 was evident
after addition of untreated ETA to the cytosolic frac-
tion. After treatment of ETA with cathepsins B and
D, EF-2 labeling was increased, especially under acidic
conditions (pH 6 > pH 5 > pH 4). However, cathep-
sin treatment of ETA in the presence of protease
inhibitors revealed [
32
P]NAD-ribose incorporation into
cytosolic EF-2 comparable to that observed in the
absence of protease treatment.
A role for mitochondria in ETA-induced cell death
has been previously shown with the use of human air-
way epithelial target cells [23]. Consequently, we exam-
ined cytochrome c release from cell-free mitochondria
isolated from control rats and then treated with ETA
in vitro (Fig. 6B, upper blots). Cytochrome c associa-
tion with intact rat liver mitochondria persisted during
the incubation in isotonic medium, despite small but
detectable release at 15 min. However, there was sub-
stantial release of cytochrome c into the resulting mito-
chondrial supernatant after the addition of native ETA
or ETA that had been pretreated with a mixture of

Cytosol
ETA-A
(37 kDa)
(66 kDa) ETA
Medium: pH 7
_
0.5 1 1.5 2 4 (h, postinjection)
ETA
Nonreducing
conditions
ETA-A
ETA-A
(37 kDa)
(66 kDa) ETA
Medium: pH 7 + ATP
ETA-A
(37 kDa)
ETA
(66 kDa)
Reducing
conditions
ETA-A
(37 kDa)
(66 kDa) ETA
Medium: pH 5
ETA-A
(37 kDa)
(66 kDa) ETA
Medium: pH 7 + ATP + Bafilomycin
A

no toxin
Native
ETA
Cathepsin-treated
ETA
EF-2
(105 kDa)
_
7654
Cathepsin-treated ETA + cytosol
4+i (pH of proteolysis)
ADP-ribosylation of EF-2
Cytochrome c
(15 kDa)
Cytochrome
c
(15 kDa)
_
515
_
515
_
5 15 (min of incubation)
Intact
mitochondria
Disrupted
mitochondria
EF-2
(105 kDa)
WB: α-EF-2

2
1
Cell death (fold stimulation)
_
5 15 30 60 90 120 240(min, postinjection)
_
0.5 1 1.5 2 4 (h, postinjection)
0.5
A
C
D
B
Fig. 6. Assessment of cytotoxic activity of cathepsin-treated ETA towards cytosolic target and mitochondria. (A) Native ETA (10 lg) was
digested in vitro for 30 min at 37 °C with a mixture of cathepsins B and D (5 UÆmL
)1
Æmg
)1
)in25mM Hepes (pH 7) or 25 mM citrate ⁄ phos-
phate buffer (pH 4–6) containing 0.1
M dithiothreitol (DT) and, when indicated, 5 lgÆmL
)1
PA and 1 lM E64 (lane 4 + i). The treated ETA
(1 lg) was then incubated for 15 min at 30 °C with the EF-2 associated with the soluble cytosolic fraction (150 lg) in 0.1
M Hepes (pH 7.4)
in the presence of 2 l
M [
32
P]NAD. Samples (20 lg) were then subjected to SDS ⁄ PAGE and analyzed by autoradiography. The dried gels
were exposed to X-ray film at )80 °C for 1–3 days. The arrow to the left indicates the mobility of
32

tions isolated from DT-injected rats (Fig. 6C, closed
bars). Finally, the kinetics and extent of production of
histone-associated DNA fragments in hepatic cytosolic
fractions following ETA administration into rats paral-
leled caspase-9 and caspase-3 activation, with DNA
fragmentation being observed 2 h after ETA injection
and remaining elevated up to 4 h (Fig. 6D, open bars).
No DNA fragmentation was observed in hepatic cyto-
solic fractions isolated from DT-injected rats (Fig. 6D,
closed bars).
Discussion
Using the in situ liver model system, we have previ-
ously shown that, after cholera toxin binding to hepa-
tic cells, cholera toxin accumulates in a low-density
endosomal compartment and then undergoes endoso-
mal proteolysis by the aspartic acid protease cathep-
sin D [22,25]. Using a similar methodology, others
have previously shown that the plant toxin ricin fol-
lows a similar intraendosomal processing pathway,
requiring ATP-dependent endosomal acidification [26].
We have recently extended these observations to DT,
and demonstrated the endosomal processing of the
internalized toxin in a sequential degradation pathway
beginning early, prior to organelle acidification via a
neutral furin activity, and followed later under acidic
conditions via cathepsin D [24]. In the present work,
we have evaluated the relationship between the
endosomal processes and cytotoxicity of ETA, another
A ⁄ B toxin functionally related to DT that has an iden-
tical intracellular target (cytosolic EF-2) [6]. Our data

determine whether ETA fragments generated by en-
dosomal cathepsins B and D fully participate in the
cytotoxic action of ETA in hepatic tissue.
Intravenously injected ETA is taken up efficiently by
the liver at an early time after death (5 min postinjec-
tion), suggesting a high binding capacity of ETA in
hepatic parenchyma. Indeed, injection of the toxin into
mice has been shown to result in an early and pro-
found inhibition of hepatic protein synthesis [27]. Our
results suggest that a
2
MG ⁄ LRP1 contributes, at least
in part, to ETA endocytosis in rat liver in vivo, based
on the following: (a) the injection of a
2
MG, which par-
tially reduced the endosomal association and process-
ing of coinjected ETA; and (b) a time-dependent
increase in immunodetectable a
2
MG ⁄ LRP1 in hepatic
endosomes induced by the toxin injection.
It has been proposed that proteolytic nicking of
ETA at the Arg279-Glu280 peptide bond mediated by
furin activity is at least partly required for expression
of ETA cytotoxicity [2,13]. In the present study, our
observation that ETA-A associates with hepatic
plasma membrane, endosomal and cytosolic fractions
isolated from ETA-injected rats is consistent with this
view. However, our in vivo and in vitro data also sup-

distinct from furin participate in ETA processing
within toxin-treated cells [31]. Moreover, additional
metallo-dependent proteolytic activities (EDTA-sensi-
tive) might act on internalized ETA within endosomes
and produce fragments with a molecular mass very
close to that of intact ETA.
All cleavages produced by cathepsins B and D in the
ETA toxin are located within ETA-A. A major degra-
dation product of ETA results from proteolytic cleav-
age at Thr396-Cys397 in the C-terminal extremity of
domain I or Ib. The degradation product contains the
entire catalytic ETA-A domain (amino acids 400–613)
extended at the N-terminus by the CPV tripeptide, and
may represent the main catalytic fragment respon-
sible for the ADP-ribosyltransferase activity identified
in vitro after cathepsin treatment. Three degradation
products (peptides 4a, 4b and 4d) displayed a molecu-
lar mass slightly less than that of the native 66 kDa
ETA and the unmodified N-terminal ETA sequence,
suggesting the removal of the C-terminal residues of
ETA encompassing the REDLK sequence. However,
an antibody that recognizes the REDLK-mediated ER
retrieval motif, which is located at the C-terminus of
ETA-A, showed immunoreactivity with endosomal
ETA-A, suggesting that the REDLK motif was not
completely lost from ETA-A within endosomes. It has
previously been shown that human serum contains a
carboxypeptidase activity, suggested to be carboxypep-
tidase-N, carboxypeptidase-H or carboxypeptidase-M,
which removed the C-terminal Lys of ETA and gener-

nelle acidification [33]. First, a low pH has been pro-
posed to be required for the proteolytic cleavage of
ETA by furin [34]. Thus, whereas furin displays an
optimal pH of  7 for model peptide substrates [35],
the proteolysis of ETA by furin is maximal between
pH 5.0 and pH 5.5 [34]. Moreover, the vacuolar
H
+
-ATPase inhibitor bafilomycin protected mouse
L cells from the toxic effects of intact ETA as well as
precleaved ETA, suggesting that an acidic environment
is required for proteolytic activation of ETA and addi-
tional event(s) leading to its cytotoxic effect [33].
Finally, it has clearly been shown that endosomal acid-
ity facilitates the binding of ETA to the endosomal
membrane of mouse L cells (maximal binding observed
at pH 4.0) and ETA-induced pore formation in the
lipid bilayer of endosomal vesicles (maximal effect at
pH < 6) [8]. Our data showing the in vitro proteolysis
of ETA by endosomal acidic cathepsins and transloca-
tion of the internalized toxin across the endosomal
membrane at low pH would be consistent with these
prior observations. Other studies reported that ETA
translocation was strictly dependent on ATP hydrolysis
but was not affected by bafilomycin, the H
+
-ATPase
inhibitor [9]. These differences may result from the
experimental approaches used (the rat liver in vivo
model versus cellular in vitro systems) and ⁄ or may be

Both pathways require the translocation of ETA into
the cytoplasm of toxin-treated cells. On the basis of
the reconstitution of the cytotoxic pathways with
in vitro cytosol and cell-free mitochondria, our data
suggest a direct interaction between ETA and cyto-
solic EF-2 on the one hand, and the mitochondrial
membrane on the other hand. However, the potential
role (if any) of ADP-ribosylation of EF-2 in the
mitochondrial apoptotic response induced by the
toxin remains to be determined. Finally, we assign
an important role to the endosomal acidic cathep-
sins B and D in increasing the in vitro transfer of
the ADP-ribosyl moiety of NAD
+
to EF-2 by ETA,
but not in the release of cytochrome c from cell-free
mitochondria.
In summary, we found that internalized ETA was
rapidly proteolyzed within rat hepatic endosomes by
cathepsins B and D, with subsequent ATP-dependent
translocation of intact ETA and ETA-A to the cytosol.
Intact ETA induced ADP-ribosylation of cytosolic
EF-2 as well as the mitochondrial release of cyto-
chrome c, both in vivo and in vitro, with the in vitro
effects being substantially increased by cathepsin B ⁄ D
pretreatment. Studies are currently underway to eluci-
date whether ETA-induced mitochondrial alteration is
mediated by the catalytic A-subunit or hydrophobic
B-domain of ETA, or whether it involves the dual het-
erogeneous part of the toxin. Use of this approach will

Cruz Biotechnology. Rabbit polyclonal antibody against
KX
5
KDEL, which recognizes the ER retention signal
KDEL and binds to various ER-resident proteins, was
obtained from S. Fuller (EMBL, Heidelberg, Germany).
Horseradish peroxidase-conjugated goat anti-(rabbit IgG)
or goat anti-(mouse IgG) were from Sigma. The protein
content of isolated fractions was determined by the method
of Lowry et al. [40]. N-terminal sequence data were
obtained by automated Edman degradation with a Procise
sequencer (Applied Biosystems, Foster City, CA, USA),
equipped with an on-line phenylthiohydantoin amino acid
analysis system. Quantitative analysis of DNA fragmenta-
tion after toxin-induced cell death was analyzed by immu-
noassay determination of cytoplasmic histone-associated
DNA fragments, according to the manufacturer’s protocol
(Roche). N-Acetyl-b-d-glucosaminidase was assayed with
p-nitrophenyl N-acetyl-b-d-glucosaminide as substrate,
according to Touster et al. [41]. Acid phosphatase was
assayed as described by Trouet [42]. Caspase-3, caspase-8
and caspase-9 activity was analyzed with a fluorometric
assay kit (BioVision, Mountain View, CA, USA) with the
respective DEVD-AFC, IETD-AFC and LEHD-AFC sub-
strates. Nitrocellulose membranes and the enhanced chemi-
luminescence detection kit were from Amersham. PA, E-64,
phenylmethanesulfonyl fluoride and EDTA were from
Sigma. HA was from Calbiochem. All other chemicals were
obtained from commercial sources and were of reagent grade.
Animals and injections

published biochemical characterizations [43,50].
Cell-free proteolysis and translocation of
endosome-associated ETA
Endosomal fractions isolated 30 min after the injection of
native ETA (15 lg per 100 g body weight) were suspended
at 1 mgÆmL
)1
in 0.15 m KCl, 5 mm MgCl
2
and 25 mm
Hepes (pH 7) or 25 mm citrate ⁄ phosphate buffer (pH 5–6)
in the presence or absence of 10 mm ATP and 0.01 lm
bafilomycin-A1. Samples were incubated at 37 °C for vari-
ous periods and subjected to reducing SDS ⁄ PAGE followed
by western blotting to determine the endosomal content
and integrity of ETA and ETA-A.
To specifically assess the membrane translocation of
intact and processed ETA through the endosomal mem-
brane, incubation mixtures were centrifuged for 60 min at
100 000 g. Pelleted endosomes and supernatants were then
subjected to reducing SDS ⁄ PAGE followed by western blot
analysis with antibody against ETA.
Cell-free translocation of mitochondria-associated
cytochrome c
A rat liver mitochondrial fraction (large-granule fraction)
was isolated by differential centrifugation as previously
described [43,46], and then resuspended at 7.5 mgÆmL
)1
in 0.15 m KCl, 5 mm MgCl
2

and proteases
The endosomal fraction ( 1 lg) was incubated for varying
lengths of time at 37 °C with 10 lg of native ETA in 30 l L
of 25 mm citrate ⁄ phosphate buffer (pH 5) or 25 mm Hepes
buffer (pH 7) containing 6 mm CaCl
2
, in the presence or
absence of protease inhibitors. To determine the integrity
of ETA, the proteolytic reaction was stopped by the addi-
tion of reducing SDS ⁄ PAGE sample buffer, and this was
followed by SDS ⁄ PAGE and western blot analysis.
For some experiments, ETA (10 lg) was digested in vitro
for varying lengths of time with bovine cathepsin B or
cathepsin D (5 UÆmL
)1
Æmg
)1
)in50mm citrate ⁄ phosphate
buffer (pH 4–6), or human furin (10 UÆmL
)1
Æmg
)1
)in
50 mm Hepes buffer (pH 7) containing 10 mm CaCl
2
and
10 mm dithiothreitol. The proteolytic reaction was stopped
by the addition of reducing SDS ⁄ PAGE buffer, and this
was followed by SDS ⁄ PAGE and Coomassie Brilliant Blue
staining or western blot analysis.

´
miramoth (Faculte
´
de Pharmacie, Chaˆ tenay-Mal-
abry, France) for assistance in the measurement of
caspase activity.
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