Role of receptor-mediated endocytosis, endosomal acidification and cathepsin D in cholera toxin cytotoxicity ´ Tatiana El Hage1,2,*, Clemence Merlen1,2*, Sylvie Fabrega1,2 and Francois Authier1,2 ¸ ˆ INSERM, U756, Chatenay-Malabry, France ˆ ´ ´ Universite Paris-Sud, Faculte de Pharmacie, Chatenay-Malabry, France Keywords acidification; cathepsin D; cholera toxin; endosome; G protein Correspondence ´ F Authier, INSERM U756, Universite Paris´ Sud, Faculte de Pharmacie, rue Jeanˆ ´ Baptiste Clement, 92296 Chatenay-Malabry, France Fax: +33 46835844 Tel: +33 46835528 E-mail: francois.authier@u-psud.fr *These authors contributed equally to this work (Received 19 December 2006, revised March 2007, accepted 20 March 2007) doi:10.1111/j.1742-4658.2007.05797.x Using the in situ liver model system, we have recently shown that, after cholera toxin binding to hepatic cells, cholera toxin accumulates in a lowdensity endosomal compartment, and then undergoes endosomal proteolysis by the aspartic acid protease cathepsin-D [Merlen C, Fayol-Messaoudi D, Fabrega S, El Hage T, Servin A, Authier F (2005) FEBS J 272, 4385– 4397] Here, we have used a subcellular fractionation approach to address the in vivo compartmentalization and cytotoxic action of cholera toxin in rat liver parenchyma Following administration of a saturating dose of cholera toxin to rats, rapid endocytosis of both cholera toxin subunits was observed, coincident with massive internalization of both the 45 kDa and 47 kDa Gsa proteins These events coincided with the endosomal recruitment of ADP-ribosylation factor proteins, especially ADP-ribosylation factor-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-ribosylation of endogenous Gsa, with maximum accumulation observed at 30–60 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 control 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 administration, 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 Cholera toxin (CT) is the causative agent of the diarrheal 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 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 2614 FEBS Journal 274 (2007) 2614–2629 ª 2007 The Authors Journal compilation ª 2007 FEBS T El Hage et al activation of cAMP-dependent protein kinase A Although the human small intestine mucosal cell is the normal target of the toxin, CT is a ubiquitous activator of adenylate cyclase in most eukaryotic cells [2] CT belongs to the AB family of bacterial exotoxins, and consists of a pentameric B subunit (CT-B) and an A subunit (CT-A) comprising two polypeptides, A1 and A2, linked by a disulfide bond CT-B binds with high affinity to GM1, a ganglioside present in apical membranes of all intestinal epithelial cells A1 has ADP-ribosyl transferase activity, whereas A2 contains a C-terminal KDEL endoplasmic reticulum (ER) retrieval signal [2] The intervening steps between CT binding and adenylate cyclase activation are not fully understood There is a characteristic lag period after CT binds to the cell surface and before an increase in adenylate cyclase activity is observed It is generally proposed that this lag period corresponds to sequential steps of CT uptake, CT activation and CT translocation to its protein target, the A subunit of the stimulatory G protein (Gsa) Two major models have been proposed to explain the events during this lag time The first model, supported by in vivo and in vitro studies on the intoxication of rat hepatocytes, suggests that CT cytotoxicity may be related, at least in part, to proteolytic events within endocytic vesicles [3–7] Following CT binding to the plasma membrane of hepatocytes, CT accumulated in a low-density endosomal compartment, with maximum accumulation observed by 15–30 [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-ribosyltransferase activity towards exogenous Gsa [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 degradation, due to its very limited number of internal lysine residues [8,9], and subsequently ADP-ribosylates Cytotoxic action of cholera toxin and cathepsin D 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 compartment(s) for CT activation [10,11] At present, no experimental data exist to support a mechanism of interaction between the active fragment(s) of CT-A generated at the endosomal locus and its target, Gsa The object of the present study was to investigate endosomally located mechanisms that regulate the activation and cytotoxic effect of CT in hepatocytes Using a subcellular fractionation approach to address the compartmentalization, activation and action of CT in vivo, we demonstrate the existence of a complex of activated CT, Gsa and ADP-ribosylation factor (ARF) protein in the endosomal membrane This coincided with ADP-ribosylation of Gsa in the endosomal compartment In addition, the aspartic acid protease inhibitor pepstatin A reduced, both in vivo and in vitro, the CTstimulated cAMP response in hepatic cells, as did transfection of MCF-7 cells with cathepsin D small interfering (si)RNA In contrast, cathepsin D overexpression in rat tumor cells increased the cAMP response to CT Finally, we report on the endosomal acidification step, which was specifically increased by CT and was required for its efficient action in rat liver and hepatoma cells Results CT-induced translocation and ADP-ribosylation of Gsa within the endolysosomal apparatus To determine whether the activated form of endosomal CT remained functional within hepatic endosomes in vivo, we first evaluated the subcellular content of Gsa (CT substrate) in endosomal fractions prepared from control and CT-injected rats (Fig 1) In agreement with our previous work [7], a time-dependent increase in CT-A and CT-B was observed in endosomal fractions 10–20 after native CT injection (Fig 1, upper left blot) or 20–90 after CT-B injection (Fig 1, upper right blot) In control rats, immunoreactive Gsa was detected as a doublet of 47 kDa and 45 kDa (Fig 1, lanes 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 (native CT; 32% increase) or 30 (CT-B; 77% increase) postinjection By 90 postinjection, both Gsa isoforms had returned to basal levels (Fig 1, lane of lower blots) FEBS Journal 274 (2007) 2614–2629 ª 2007 The Authors Journal compilation ª 2007 FEBS 2615 Cytotoxic action of cholera toxin and cathepsin D T El Hage et al Fig 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 scanning densitometry, with results expressed as percentage of signal intensity in the endosomal fraction prepared from control (noninjected) rats 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 prepared 20 after CT administration (Fig 2A) When LPS fractions were incubated at °C, most of the CTB and Gsa appeared in a single broad region with a density of 1.077–1.119 gỈmL)1 (Fig 2A, left blots), which mainly coincided with the Golgi marker galactosyltransferase (Fig 2A, upper left panel) and the endosomal marker EEA1 or procathepsin D precursor (Fig 2B) When the LPS fraction was incubated at 37 °C, there were only minor changes in the distribution of CT-B (Fig 2A, upper right blot), with a slight shift to the right that partially coincided with the lysosomal marker acid phosphatase (Fig 2A, upper right panel) and the mature 45 kDa cathepsin D enzyme (Fig 2B, lower blot) This was accompanied by a partial loss in CT-B immunoreactivity at the endosomal position (Fig 2A, upper right blot) However, a major 2616 transfer of both Gsa proteins from the endosomal to the lysosomal position was clearly detectable, along with a partial decrease in the total amount of immunoreactive Gsa throughout the gradient (Fig 2A, lower right blot) The cofactor ARF, and especially ARF-6, is required for full ADP-ribosylation of Gsa by activated CT [12] Therefore, we evaluated the subcellular content of ARF proteins in hepatic fractions prepared from CT and CT-B-injected rats (Fig 3A) An increase in ARF content was observed in endosomal fractions isolated postinjection of CT-B, and this increase was maintained for up to 60 (Fig 3A, upper panel) CT administration led to a low and brief recruitment of ARF-6 to the endosomal membrane 15–30 postinjection (Fig 3A, lower panel EN), a decrease in plasma membrane ARF-6 content 5–15 postinjection (Fig 3A, panel PM), and a sustained association of ARF-6 with the cytosolic fraction 5–60 postinjection (Fig 3A, panel S) Finally, we performed an in vivo CT substrate labeling experiment using [32P]NAD and endocytic vesicles that contained in vivo internalized native FEBS Journal 274 (2007) 2614–2629 ª 2007 The Authors Journal compilation ª 2007 FEBS T El Hage et al Cytotoxic action of cholera toxin and cathepsin D A B Fig Transfer of CT-B and Gsa from the endosomal to the lysosomal position on Nycodenz gradients (A) The LPS fraction was isolated 20 after CT administration, and immediately subfractionated on linear Nycodenz density gradients (left panels, °C), or incubated with ATP and an ATP-regenerating system at 37 °C for 60–90 prior to subfractionation on linear Nycodenz density gradients (right panels, 37 °C) Galactosyltransferase (circles) and acid phosphatase (squares) activities were determined, and results expressed as a percentage of total enzymatic activity recovered CT subunits and Gsa content were evaluated for each subfraction by immunoblotting Thirty microliters of each subfraction was loaded onto each lane Arrowheads indicate the median densities of galactosyltransferase (closed arrowhead) and acid phosphatase (open arrowhead) Arrows on the right indicate the mobilities of immunodetected CT-B ( 12 kDa) and Gsa ( 47 and 45 kDa) CT-A was below the limits of detection (results not shown) (B) The content of early endosome antigen (EEA1) and cathepsin D (CD) was evaluated by immunoblotting for each subfraction isolated from the LPS fraction incubated at 37 °C Components appearing at densities 1.075–1.105 and 1.11–1.14 gỈmL)1 were scored, respectively, as truly endosomal and lysosomal CT (Fig 3B) Radiolabeling of endosomal Gsa was observed with endosomal fractions prepared 30 and 60 postinjection of native CT Thus, CT is active in vivo towards endosomal Gsa following a 30 lag period, which probably corresponds 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 endosomes displayed increased rates of acidification and a more acidic steady-state intravesicular pH [13] Therefore, we investigated whether CT altered endosomal FEBS Journal 274 (2007) 2614–2629 ª 2007 The Authors Journal compilation ª 2007 FEBS 2617 Cytotoxic action of cholera toxin and cathepsin D T El Hage et al 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 with bafilomycin A1 alone (0.2 lm) abolished the granular fluorescence of DAMP almost completely (Fig 4B, lower left panel) However, a residual fluorescent staining reminiscent of vesicular acidification was clearly observed in cells pretreated with bafilomycin A1 and then incubated with CT for h (Fig 4B, lower right panel) These data are consistent with our finding that CT increased endosomal acidification at the early stage of CT action A B Role of endosomal acidification and cathepsin D in CT action Fig CT-mediated recruitment of ARF-6 and ADP-ribosylation of Gsa in hepatic endosomes (A) Rat liver endosomal (EN), plasma membrane (PM) and cytosolic (S) 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 ARF and ARF-6 using their respective polyclonal and monoclonal antibodies 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 lM [32P]NAD at 30 °C in an ADP-ribosylation buffer; this was followed by SDS ⁄ PAGE and autoradiography Molecular mass markers are indicated to the left of the panel The arrow on the right indicates the mobility of [32P]-labeled Gsa ( 45 kDa) acidification during the early stage of CT action, when most of the internalized CT should be located within hepatic endosomes We used a fluorescent weak base, acridine orange, which concentrates within acidic compartments and has been widely used to assess vacuolar H+-ATPase activity [14,15] A time-dependent decrease in fluorescence intensity was observed within hepatic endosomes prepared from uninjected rats as well as toxin-injected rats, both with (closed symbols) and without (open symbols) addition of ATP (Fig 4A) However, endosome acidification was strongly ATP-dependent, and, in the presence of ATP, the rate of acidification was markedly increased following CT administration (closed squares) The initial rate of ATP-dependent acidification of endosomes (which was linearly related to incubation time for the first 2618 To assess whether the aspartic acid protease cathepsin 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 intraperitoneal injection of either pepstatin A, an inhibitor of aspartic acid proteases [17], or a mixture of bafilomycin A1 and folimycin, two inhibitors of the vacuolar ATPases [16], prior to CT administration (50 lg per 100 g body weight) Rats were then killed 50 post-CT injection The cAMP content in rat liver homogenates isolated from control rats was increased  5-fold over basal levels after CT injection (Fig 5A, cf Basal and CT-Control) Both pepstatin A and bafilomycin A1 ⁄ folimycin treatment caused a  3-fold decrease in hepatic cAMP content in CT-treated rats (Fig 5A, cf PA, Bafi ⁄ Foli and Control) Cellular cAMP content was next measured in vitro in hepatoma HepG2 cells treated with CT in the presence or absence of pepstatin A or bafilomycin A1 (Fig 5B) Cellular cAMP content increased 45 after the addition of CT, and reached a maximum 90–120 (closed circles) Bafilomycin A1 (closed triangles) extended the lag phase by 15 min, and decreased the rate at which CT increased cellular cAMP content Pepstatin A (closed squares) was less effective at inhibiting the initial rate of cAMP production, but did reduce the maximal extent of cAMP production FEBS Journal 274 (2007) 2614–2629 ª 2007 The Authors Journal compilation ª 2007 FEBS T El Hage et al Cytotoxic action of cholera toxin and cathepsin D Fluorescence Intensity (A.U.) A B Basal Cholera toxin H 400 No drugs 200 control control + ATP cholera toxin cholera toxin + ATP CT-B subunit + ATP diphtheria toxin diphtheria toxin + ATP 15 Time of incubation (min) Baf-A1 30 Fig Effect of cholera toxin on endosomal acidification (A) Rat liver endosomal fractions were isolated h after the in vivo administration of native CT, CT-B or diphtheria toxin (60 lg per 100 g body weight), and incubated in 0.15 M KCl containing mM MgCl2, lM acridine orange and, when indicated, mM ATP The relative decrease in fluorescence intensity was immediately recorded at 37 °C for 30 using a recording spectrofluorometer Results are expressed as arbitrary units of fluorescence intensity Baseline fluorescence at zero time was 284.63 ± 7.41 (– ATP) and 422.97 ± 1.79 (+ ATP) (B) HepG2 hepatoma cells were incubated at 37 °C for 30 with or without bafilomycin A1 (0.2 lM), and this was followed by the addition of CT (1.3 lM) or buffer alone for an additional h The acidic compartments were visualized by immunofluorescence using the DAMP method Fig Role of vesicular acidification and cathepsin D in cellular cAMP production by CT (A) Rats were injected with native CT (50 lg per 100 g body weight) h after an intraperitoneal injection of either 12.5% dimethylsulfoxide, 625 lg pepstatin A methyl ester (PA) or a mixture of bafilomycin A1 ⁄ folimycin (Bafi ⁄ Foli) (0.75 lg each) Fifty minutes after CT injection, rats were killed, hepatic homogenates were prepared, and cAMP content was measured by radioimmunoassay The data were expressed as pmol cAMP per mG protein Each histogram represents the mean ± SD of at least three independent determinations (B) HepG2 hepatoma cells were treated with CT (10 lgỈmL)1) and incubated at 37 °C in the absence (control) or presence of pepstatin A (120 lgỈmL)1) or bafilomycin A1 (0.2 lM) for the indicated times Cellular cAMP content was measured as described for (A), and the data were expressed as pmolỈ(mG protein))1 Results are the mean ± SD of three separate experiments FEBS Journal 274 (2007) 2614–2629 ª 2007 The Authors Journal compilation ª 2007 FEBS 2619 Cytotoxic action of cholera toxin and cathepsin D T El Hage et al A B Fig Relationship between cathepsin D expression and cellular cAMP response to CT (A) Rat embryonic 3Y1-Ad12 tumor cells expressing either no cathepsin D (control cells, open histograms) or overexpressing human wild-type cathepsin D (3Y1-Ad12-CD, closed histograms) were incubated with CT (1.3 lM) for h Cellular cAMP content was measured, and expressed as fold stimulation over basal (unstimulated) activity [6 pmolỈ(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) (B) MCF-7 cells, whose cathepsin D expression was inhibited by siRNA silencing for 48–72 h, were incubated with CT (1.3 lM) for h Cellular cAMP content was measured and expressed as fold stimulation over basal (unstimulated) activity [ 28 pmolỈ(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) 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 cytotoxicity 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 cells (Fig 6A, lower panel) Measurement of cellular cAMP levels in 3Y1-Ad12 cells after CT treatment revealed that cells expressing the transfected gene were  3–5-fold more sensitive to CT treatment than were control cells deficient in cathepsin D (Fig 6A, 2620 upper panel) To strengthen the possibility of a direct action of cathepsin D in CT cytotoxicity, cAMP assays were performed with MCF-7 cells whose endogenous cathepsin D expression was inhibited by small RNA-mediated gene silencing (cathepsin D siRNA) (Fig 6B) A progressive decrease in expression of both procathepsin D and mature cathepsin D was observed in MCF-7 cells 48–72 h after transfection with cathepsin D siRNA (Fig 6B, lower panel) On the basis of cellular cAMP levels, transfected MCF-7 cells were  4-fold less effective in responding to CT treatment as compared to nontransfected MCF-7 cells (Fig 6B, upper panel), supporting the notion that cathepsin D might play a crucial role in CT activation and action in hepatic cells as well as in other cell types FEBS Journal 274 (2007) 2614–2629 ª 2007 The Authors Journal compilation ª 2007 FEBS T El Hage et al Cytotoxic action of cholera toxin and cathepsin D KDEL peptide in the internalized CT-A No perceptible KDEL immunoreactivity was detected in rat liver endosomal fractions isolated 5–90 post-CT injection (Fig 7B), suggesting that the integrity of the C-terminal KDEL peptide was rapidly lost during CT endocytosis We have previously identified a 25 kDa endosomal CT-A fragment, which we postulated might be involved in the ADP-ribosylation of Gsa [7] The 25 kDa fragment was consistently observed in endosomal hydrolysates of CT obtained at acidic pH, and was strictly cathepsin D-dependent; its detection coincided with 32P-labeling of Gsa by CT hydrolysates at acidic pH [7] Consequently, we examined whether, under conditions where a 25 kDa CT-A fragment was generated by endosomal cathepsin D, we would observe a corresponding loss of the C-terminal KDEL peptide (Fig 7C) Indeed, whereas rapid production of 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 fragment(s) that are active towards Gsa 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 A CT CT-A CT CT-A CT CT-A CT B CT-A CT CT-A (28-kDa) 15 30 60 90 CT 15 30 60 90 CT-A (28-kDa) CT-B (12-kDa) CT-B (12-kDa) α-CT α-KSEKKDEL C α-KX5KDEL – – 30 α-CT α-KAVKKDEL 30 – – α-KAVKKDEL pH 60 Time of incubation (min) CT-A (28-kDa) CT-B (12-kDa) α-CT α-KAVKKDEL Fig 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 lg of CT-A or 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 antiserum 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 mM 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 are indicated on the left (CT-A,  28 kDa; CT-B,  12 kDa) A major 25 kDa CT-A fragment was evident after 30 of incubation, but was not recognized by the antibody to KDEL FEBS Journal 274 (2007) 2614–2629 ª 2007 The Authors Journal compilation ª 2007 FEBS 2621 Cytotoxic action of cholera toxin and cathepsin D T El Hage et al a 25 kDa product of CT-A was evident following proteolysis of CT at acidic pH (Fig 7C, left panel), no detectable KDEL immunoreactivity was associated with this degradative CT-A product (Fig 7C, right panel) Discussion In the present study, we show that rat hepatocytes display endosomally located mechanisms to regulate CT activation and action Two of these regulatory mechanisms may be at the level of intraendosomal toxin proteolysis and endosome acidification (Fig 8) The assessment of CT compartmentalization during its endocytosis into rat liver has revealed specific in vivo regulation during the early phase (0–60 min) of toxin internalization First, a complex of activated CT-A, cointernalized Gsa and recruited ARF proteins was observed in endosomes 5–30 postinjection Second, efficient ADP-ribosylation of the cointernalized Gsa proteins occurred at the endosomal locus after a lag phase of 30 Third, CT-mediated hyperacidifi- cation of endosomes increased over a time course similar to that of endosomal activation of CT Therefore, we propose that endosome regulation would serve as an effective amplification mechanism for promoting CT and cathepsin D interaction to induce a maximal cytotoxic effect (Fig 8) Finally, using in vitro cellular systems, we have obtained evidence that the cAMP response in CT-treated cells was, at least in part, related to the proteolytic activity and expression level of the aspartic acid protease cathepsin D (Fig 8) However, 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 mediators 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 Endosomal regulation of CT activation and action in rat liver 2622 FEBS Journal 274 (2007) 2614–2629 ª 2007 The Authors Journal compilation ª 2007 FEBS T El Hage et al 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 fractions isolated from noninjected rats These isoforms are produced by alternative splicing of a single precursor mRNA [23] The translocation of G proteins from the plasma membrane to the endosomal apparatus has been demonstrated biochemically and morphologically in various cells stimulated by agonists such as glucagon [21], isoproterenol [24], carbachol [25], thyrotropin-releasing hormone [26] and bradykinin [27] Our studies extend these observations to CT, for which maximal endosomal association of Gsa was observed 20 (native CT) or 30 (CT-B) postinjection The underlying mechanisms involved in CT-induced endosomal translocation of Gsa may well originate in target lipid rafts at the cell surface, which show significant enrichment of stimulatory and inhibitory G protein and predominant localization of the endogenous CT receptor ganglioside GM1 [28,29] This would facilitate interactions between CT, GM1 and Gsa and, potentially, their subsequent cointernalization Alternatively CT, which increases the endosomal content of fluid-phase endocytosis probes in rat liver [30], might induce the uptake of various plasma membrane molecules, such as its own cellular protein target Gsa, into the endocytic pathway Our data suggest a direct interaction between activated CT-A and Gsa in the endosomal membrane It has clearly been shown that the endosomal acidic pH facilitates the membrane insertion and penetration of intact CT-A and ⁄ or activated CT-A fragment(s) across the endosomal membrane Using the lipid bilayer matrix containing ganglioside GM1, fluorescence and phosphorescence spectroscopy studies have shown that upon CT binding to GM1, CT-A faces the membrane surface but does not significantly penetrate into the hydrophobic core of the bilayer at neutral pH [31,32] However, CT-A1 peptide released from CT-A2 peptide exhibits hydrophobic behavior in aqueous solution and when membrane-bound, suggesting that free CT-A1 peptide or CT-A fragment may partition spontaneously 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 capable 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 Cytotoxic action of cholera toxin and cathepsin D 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 markedly increases endosomal acidification rates (this study) [13], to allow maximal insertion of activated CT-A into the endosomal membrane, leading to efficient ADPribosylation of cointernalized Gsa The enzymatic activity of CT-A1 is allosterically stimulated by ARFs, which are host-cell small GTPbinding proteins active in the GTP-bound form [34] On the basis of the reconstitution of a signal transduction 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 human ARF-6 [35] However, inhibition of the ARF-6 pathway had minimal effects on CT entry, intracellular CT transport, and CT-induced activation of adenylate cyclase [36] Although the CT–ARF interaction has been extensively characterized in vitro, little is known about their in vivo interaction, and the subcellular binding site(s) between CT and ARF-6 remain(s) undefined Originally, ARF-6 was thought to be an unconventional member of the ARF family that was found exclusively in the plasma membrane of Chinese hamster ovary cells [37] However, assessment of the subcellular distribution of endogenous ARF-6 in various other tissues and cells has established both a cytosolic and membrane-bound localization [38], and overexpressed ARF-6 has been localized to the plasma membrane and endosomes [39] In the present study, we demonstrate the existence of an endosomal pool of ARFs whose amount was strongly increased after CT treatment ARF-6 was undetectable in endosomes prepared from untreated rats, but its endosomal recruitment was rapidly observed after CT injection Recently, it was shown that V-ATPase-dependent endosomal acidification stimulates the recruitment of ARF-6 from proximal tubule cytosol to endosomal membranes, implicating this process in endosomal function in situ [40] However, the precise functional role of endosomal ARF-6 in the full activation of internalized CT-A remains to be determined Subcellular fractionation techniques used to assess the in vivo localization of [125I]CT uptake into rat liver have previously shown that some radioactivity (30 to h postinjection) is intrinsic to acid-phosphatasecontaining structures, presumably lysosomes [4] Using the in situ rat liver model system for endosome– lysosome fusion, we have confirmed a low lysosomal FEBS Journal 274 (2007) 2614–2629 ª 2007 The Authors Journal compilation ª 2007 FEBS 2623 Cytotoxic action of cholera toxin and cathepsin D T El Hage et al transfer of internalized native CT-B, which was accompanied by a sustained transfer of Gsa, the CT substrate The endolysosomal transfer of CT-B was also accompanied by a net decrease in its immunoreactivity throughout the gradient, suggesting that CT-B is proteolyzed 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 suggests 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 to an increase in the lag phase for activation of adenylate cyclase by CT and a decrease of 3–10-fold in the apparent affinity of the toxin for the enzyme [6] Moreover, both chloroquine and monensin interfered with the ATP-dependent intraendosomal degradation of internalized radioactive CT [6] Recently, we have identified an endosomal aspartic acid protease, cathepsin D, that binds specifically to endocytosed CT at acidic pH and generates degradative CT fragments displaying ADP-ribosyltransferase activity towards exogenous Gsa [7] This would suggest that within hepatocytes, vacuolar acidification participates in the activation and action of CT, and our present study is consistent with this view Considering the nonacidotropic effects of chloroquine [41], we showed that bafilomycin, a macrolide antibiotic that specifically inhibits vacuolar ATPase at concentrations up to lm, similarly affected CT action in hepatocytes This decrease in vacuolar acidification was accompanied both in vivo and in vitro by a corresponding reduction in the cAMP response of hepatic cells to CT Finally, we have shown here that expression of cathepsin D in the cathepsin D-deficient 3Y1-Ad12 cell line led to an increase in CT action, whereas siRNA of cathepsin D in MCF-7 cells reduced CT sensitivity Our data, together with previous studies [7], assign an important role to endosomal acidic cathepsin D in promoting the cytotoxic action of CT, most probably through the removal of C-terminal residues of CT-A1 encompassing the terminal KDEL peptide Our in vivo and in vitro studies with hepatocytes are in marked contrast to in vitro studies using other cellular 2624 models, where acidotropic drugs and V-ATPase inhibitors 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 endocytosis: 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 predominantly altered the earlier steps of endocytosis [44] However, 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 endosomal acidification, which increased two-fold after CT treatment, compared favorably with these previous reports [13,30] Moreover, we provide three supplemental observations: (a) endosomal acidification rates were significantly increased h post-CT treatment, which coincided with the presence of internalized CT within endocytic vesicles; (b) endosome acidification rates were unaffected by diphtheria toxin and free CT-B, which confirmed the potential role of cAMP in mediating the effects of CT on endosome acidification [13]; and (c) the effects of CT were not observed when endosome acidification was measured in the absence of ATP, possibly due to a direct effect(s) of CT and ⁄ or cAMP on the activity and ⁄ or abundance of the endosomal H+ pump The effects of CT on endosomal acidification are likely to be mediated, at least in part, by increased intracellular cAMP Thus, the effects of CT were reproduced by direct administration to perfused livers of dibutyryl cAMP [13] or by intraperitoneal injection of pertussis toxin to rats, which increased liver cAMP significantly [13] In contrast, in vivo administration of diphtheria toxin (which does not modify the intracellular cAMP level) or CT-B (for which cytotoxic activity is nonexistent) had no effect on endosome acidification Also, it is possible that CT causes an activation of the vacuolar H+-pump and ⁄ or Cl– transport, as well as changes in the remodeling and maturation of early endocytic vesicles in response to cAMP [13] Finally, our findings would be consistent with a model in which CT induced an increase in the 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 suggest that the more acidic pH of endocytic vesicles at FEBS Journal 274 (2007) 2614–2629 ª 2007 The Authors Journal compilation ª 2007 FEBS T El Hage et al an early stage may be part of the toxic action of CT, facilitating the proteolytic activation of CT-A by cathepsin 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 CT-A and CT-B, native CT, acridine orange and pepstatin A were purchased from Sigma (St Louis, MO, USA) A nontoxic diphtheria toxin CRM 197 mutant and bafilomycin-A1 were obtained from Calbiochem (San Diego, CA, USA) Rabbit polyclonal anti-(CT C3062) was obtained from Sigma Rabbit polyclonal IgG directed against Gsa proteins was obtained from NEN Rabbit anti-(mouse cathepsin D R291) [7] was obtained from J S Mort (Shriners Hospital for Crippled Children, Montreal, Quebec, Canada) Rabbit polyclonal IgG broadly reactive with ARF family proteins, mouse monoclonal IgG directed against full-length ARF-6 and goat polyclonal IgG directed against the C-terminus of rat cathepsin D were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA) Mouse monoclonal antibody directed against the rat early endosome antigen (EEA1) was purchased from Transduction Laboratories Rabbit polyclonal antibodies against KAVKKDEL, KSEKKDEL or KXXXXXKDEL, which recognize the ER retention signal KDEL and bind to various ER-resident proteins, were obtained from S Fuller (EMBL, Heidelberg, Germany) Fluorescein isothiocyanateconjugated rabbit polyclonal IgG to dinitrophenyl-KLH was obtained from Molecular Probes Horseradish peroxidase-conjugated goat anti-(rabbit) IgG and anti-(mouse) IgG were obtained from Bio-Rad (Hercules, CA, USA) The protein content of isolated fractions was determined by the method of Lowry et al [45] Galactosyltransferase was assayed as described by Beaufay et al [46] Acid phosphatase was assayed as described by Trouet [47] Nitrocellulose membranes and Enhanced ChemiLuminescence detection 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 Cytotoxic action of cholera toxin and cathepsin D Animals and injections In vivo procedures were approved by the institutional committee for use and care of experimental animals Male Sprague-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 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 centrifugation, 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 mm sodium phosphate (pH 7.4), disrupted in the same hypotonic medium using a small Dounce homogenizer (15 strokes with a Type A pestle), and centrifuged at 150 000 g for 60 in a Beckman 70.1 Ti rotor as previously described [7,17,48–50] In vitro endosome–lysosome transfer reaction The cell-free endosome–lysosome transfer of in vivo internalized CT and Gsa was performed as described previously [21,52,53] The postmitochondrial supernatant (referred to as the LPS fraction) was incubated for 60–90 at °C or 37 °C with mm ATP, mgỈmL)1 creatine kinase, and 20 mm phosphocreatine After cooling to °C, incubation mixtures were subjected to centrifugation on linear Nycodenz gradients at 200 000 g for 60 in a Beckman SW41 rotor as described previously [21,52,53] The distribution of CT, Gsa and enzyme activities was determined, and components appearing at densities 1.065–1.11 and 1.11–1.145 gỈmL)1 were scored as endosomal and lysosomal, respectively Immunoblot analysis Electrophoresed samples were transferred onto nitrocellulose membranes for 60 at 380 mA in transfer buffer containing 25 mm Tris base and 192 mm glycine The membranes were blocked by a h incubation with 5% skimmed milk in 10 mm Tris ⁄ HCl (pH 7.5), 300 mm NaCl, and 0.05% Tween-20 The membranes were then incubated with primary antibody [rabbit polyclonal serum against native FEBS Journal 274 (2007) 2614–2629 ª 2007 The Authors Journal compilation ª 2007 FEBS 2625 Cytotoxic action of cholera toxin and cathepsin D T El Hage et al CT C3062 (diluted : 60 000), KAVKKDEL (diluted : 100), KSEKKDEL (diluted : 100), KXXXXXKDEL (diluted : 100), affinity-purified rabbit polyclonal IgG against human ARF-1 (diluted : 400) and Gsa (diluted : 10 000) and mouse monoclonal IgG against human ARF-6 (diluted : 100)] in the above buffer for 16 h at °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 h at room temperature 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 [32P]NAD, 50 mm sodium phosphate buffer (pH 7.2), 0.5 mm GTP, mm ATP, mm MgCl2, and 10 mm thymidine, and incubated at 30 °C for 45 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 For other experiments, hepatoma HepG2 cells were preincubated with mm isobutylmethylxanthine for 30 The medium was then supplemented with CT (10 lgỈmL)1) and incubated for 15 to h with or without pepstatin A methyl ester (120 lgỈmL)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 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% CO2 [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% CO2 [56] Cathepsin D-transfected 3Y1-Ad12 carcinoma cells were grown in RPMI medium supplemented with 5% fetal bovine serum, 400 lgỈmL)1 G418 and 50 lgỈmL)1 gentamicin in an atmosphere of 95% air ⁄ 5% CO2 [18] Transfection and RNA interference ATP-dependent acidification of isolated endosomes was assayed using acridine orange, a membrane-permeable lipophilic 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, mm MgCl2, lm acridine orange and, when indicated, mm ATP These suspensions were immediately placed in the spectrofluorometer, and fluorescence intensities were recorded at 37 °C for 1–30 Cathepsin D siRNA and control siRNA were synthesized by Eurogentec (Seraing, Belgium) The siRNA sequence for human cathepsin D has been previously described [57] MCF-7 cells were grown in six-well plates for days, and then transiently transfected with lg of siRNA using lL of Oligofectamine (Invitrogen, Carlsbad, CA, USA) Cells were then incubated for h at 37 °C prior to the addition of 5% fetal bovine serum Cells were harvested 48–72 h post-transfection, and subjected to western blot analysis (to monitor cathepsin D expression levels) and cAMP assays cAMP assays Immunofluorescence Following injection of native CT, rats were sacrificed at 50 and livers were rapidly removed and minced in cold ethanol containing mm isobutylmethylxanthine (40 mL per liver) For some experiments, rats received an intraperitoneal injection of pepstatin A methyl ester (62.5 lg), bafilomycin-A1 ⁄ folimycin (0.75 lg each) or solvent (dimethylsulfoxide) h prior to injection of CT Livers were homogenized on ice for using a Polytron, frozen in liquid N2, thawed at 21 °C three times, and then centrifuged at 25 000 g for 20 at °C Supernatants were dried using a Speedvac and then dissolved in cAMP assay buffer The cAMP present in the samples was measured by radioimmunoassay as previously described [13] Protein concentration was determined in parallel, and data were expressed as pmol cAMP per mG protein The acidic compartments in hepatoma HepG2 cells were visualized by immunofluorescence using the DAMP method [41] Cells grown on glass coverslips were washed twice with NaCl ⁄ Pi before incubation at 37 °C for 30 with serumfree DMEM in the presence or absence of 0.2 lm bafilomycin A1 Cells were then treated with CT (1.3 lm) or buffer alone for h, and this was followed by the addition of DAMP (30 lm) for 30 Cells were then washed three times with NaCl ⁄ Pi, and fixed with 3% paraformaldehyde in NaCl ⁄ Pi for 20 Fixed cells were treated with 50 mm NH4Cl for 15 min, washed with NaCl ⁄ Pi, and permeabilized with 0.1% Triton X-100 in NaCl ⁄ Pi for and 0.5% saponine in NaCl ⁄ Pi for 10 Permeabilized cells were blocked for 20 with 10% horse serum in NaCl ⁄ Pi, and then incubated for 60 with a rabbit polyclonal anti-dinitrophenyl fluorescein conjugate (1 : 100 dilution) 2626 FEBS Journal 274 (2007) 2614–2629 ª 2007 The Authors Journal compilation ª 2007 FEBS T El Hage et al Laser-scanning confocal microscopy was performed using a Zeiss LSM 510 confocal (Axiovert 100 m) inverted microscope equiped with a Zeiss X63 ⁄ 1.4 NA oil immersion objective lens (plan-Apochromat) Cytotoxic action of cholera toxin and cathepsin D 10 Acknowledgements We thank Pamela H Cameron (McGill University, Montreal, Quebec, Canada) for reviewing the manuscript and assistance in these 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Authier F, Di Guglielmo GM, Bergeron JJM & Brodt P (2001) Inhibition of endosomal insulin-like growth factor-I processing by cysteine proteinase inhibitors blocks receptor-mediated functions J Biol Chem 276, 13644–13649 ´ 57 Laurent-Matha V, Maruani-Herrmann S, Prebois C, Beaujouin M, Glondu M, Noel A, Alvarez-Gonzalez ă ML, Blacher S, Coopman P, Baghdiguian S et al (2005) Catalytically inactive human cathepsin D triggers fibroblast invasive growth J Cell Biol 168, 489–499 FEBS Journal 274 (2007) 2614–2629 ª 2007 The Authors Journal compilation ª 2007 FEBS 2629 ... chloroquine and monensin interfered with the ATP-dependent intraendosomal degradation of internalized radioactive CT [6] Recently, we have identified an endosomal aspartic acid protease, cathepsin D, ... action of cholera toxin and cathepsin D T El Hage et al min) increased two-fold in endosomes isolated from CT-injected rats (closed squares), but this was not observed for CT-B-injected (closed diamonds)... CT action A B Role of endosomal acidification and cathepsin D in CT action Fig CT-mediated recruitment of ARF-6 and ADP-ribosylation of Gsa in hepatic endosomes (A) Rat liver endosomal (EN), plasma