Proteolytic activation of internalized cholera toxin within hepatic endosomes by cathepsin D

We have defined the in vivo and in vitro metabolic fate of internalized cholera toxin (CT) in the endosomal apparatus of rat liver. In vivo, CT was internalized and accumulated in endosomes where it underwent degradation in a pH-dependent manner. In vitro proteolysis of CT using an endosomal lysate required an acidic pH and was sensitive to pepstatin A, an inhibitor of aspartic acid proteases. By nondenaturating immunoprecipitation, the acidic CT-degrading activity was attributed to the luminal form of endosomal cathepsin D. The rate of toxin hydrolysis using an endosomal lysate or pure cathepsin D was found to be high for native CT and free CT-B subunit, and low for free CT-A subunit. On the basis of IC(50) values, competition studies revealed that CT-A and CT-B subunits share a common binding site on the cathepsin D enzyme, with native CT and free CT-B subunit displaying the highest affinity for the protease. By immunofluorescence, partial colocalization of internalized CT with cathepsin D was confirmed at early times of endocytosis in both hepatoma HepG2 and intestinal Caco-2 cells. Hydrolysates of CT generated at low pH by bovine cathepsin D displayed ADP-ribosyltransferase activity towards exogenous Gsalpha protein suggesting that CT cytotoxicity, at least in part, may be related to proteolytic events within endocytic vesicles. Together, these data identify the endocytic apparatus as a critical subcellular site for the accumulation and proteolytic degradation of endocytosed CT, and define endosomal cathepsin D an enzyme potentially responsible for CT cytotoxic activation.     

Proteolytic cleavage of ricin A chain in endosomal vesicles. Evidence for the action of endosomal proteases at both neutral and acidic pH

Macrophages actively internalize macromolecules into endosomal vesicles containing proteases. The plant toxin, ricin A chain delivered into this pathway by receptor-mediated endocytosis, was found to be exquisitely sensitive to cleavage by these proteases. Proteolytic fragments of ricin A chain were generated within cells as early as 2-3 min after internalization. Toxin proteolysis was initiated in early endosomal vesicles, and transport to lysosomes was not required. As endosomes transit the cell, their lumenal pH drops from neutral to acidic. Previous studies in macrophages had suggested that endosomal proteolysis is dependent on vesicle acidification. Isolated endosomal vesicles containing ricin A chain catalyzed the cleavage of this protein in vitro; however, proteolysis was observed at both neutral and acidic pH. Experiments using isolated endosomes demonstrated that both cysteine and aspartyl proteases were responsible for the cleavage of ricin A chain. The cysteine protease, cathepsin B, catalyzed toxin proteolysis in endosomes between pH 4.5 and 7.0 while aspartyl protease activity was maximal below pH 5.5. Radiolabeling the lumenal contents of macrophage endosomes confirmed that both the cysteine protease, cathepsin B, and the aspartyl protease, cathepsin D, were present in these vesicles. These proteases were not present on the plasma membrane but were found in early endosomes indicating they are derived from an intracellular source. The presence of proteases with different pH optima in early endosomes suggests that processing in these vesicles may be regulated by changes in endosomal pH. This result represents an important difference in protein processing in endosomes versus lysosomes and provides new insights into the function of endosomal proteases.     

Endosomal proteolysis of insulin-like growth factor-I at its C-terminal D-domain by cathepsin B

IGF-I is degraded within the endosomal apparatus as a consequence of receptor-mediated endocytosis. However, the nature of the responsible protease and the position of the cleavage sites in the IGF-I molecule remain undefined. In vitro proteolysis of IGF-I using an endosomal lysate required an acidic pH and was sensitive to CA074, an inhibitor of the cathepsin B enzyme. By nondenaturing immunoprecipitation, the acidic IGF-I-degrading activity was attributed to the luminal species of endosomal cathepsin B with apparent molecular masses of 32- and 28-kDa. The cathepsin B precursor, procathepsin B, was processed in vitro within isolated endosomes at pH 5 or at 7 in the presence of ATP, the substrate of the vacuolar H(+)-ATPase. The rate of IGF-I hydrolysis using an endosomal lysate or pure cathepsin B was found to be optimal at pH 5-6 and moderate at pH 4 and 7. Competition studies revealed that EGF and IGF-I share a common binding site on the cathepsin B enzyme, with native IGF-I displaying the lowest affinity for the protease (IC50 approximately 1.5 microM). Hydrolysates of IGF-I generated at low pH by endosomal IGF-I-degrading activity and analyzed by reverse-phase HPLC and mass spectrometry revealed cleavage sites at Lys68-Ser69, Ala67-Lys68, Pro66-Ala67 and Lys65-Pro66 within the C-terminal D-domain of IGF-I. Treatment of human HepG2 hepatoma cells with the cathepsin B proinhibitor CA074-Me reduced, in vivo, the intracellular degradation of internalized [125I]IGF-I and, in vitro, the degradation of exogenous [125I]IGF-I incubated with the cell-lysates at pH 5. Inhibitors of cathepsin B and pro-cathepsin B processing, which abolish endosomal proteolysis of IGF-I and alter tumor cell growth and IGF-I receptor signalling, merit investigation as antimetastatic drugs.     

Role of receptor-mediated endocytosis, endosomal acidification and cathepsin D in cholera toxin cytotoxicity

Using the in situ liver model system, we have recently shown that, after cholera toxin binding to hepatic cells, cholera toxin accumulates in a low-density 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 J272, 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 Gsalpha 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 (Gsalpha) translocation. After an initial lag phase of 30 min, these constituents were linked to NAD-dependent ADP-ribosylation of endogenous Gsalpha, with maximum accumulation observed at 30-60 min postinjection. Assessment of the subsequent postendosomal fate of internalized Gsalpha revealed sustained endolysosomal transfer of the two Gsalpha 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.     

Tetanus toxin induces fusion and aggregation of lipid vesicles containing phosphatidylinositol at low pH

We report here on the ability of tetanus toxin to induce, at low pH, fusion and aggregation of lipid vesicles containing phosphatidylinositol. It has been shown that diphtheria toxin is internalized in acidic vacuoles (endosomes) and that the low endosomal pH could induce a protein conformational change responsible for the interaction with the endosomal membranes and the toxin translocation into the cytoplasm. The data here reported indicate that tetanus toxin might interact with lipid membrane in a similar way as diphtheria toxin suggesting for the two proteins an identical mechanism of entry into cells.     

Selective translocation of the A chain of diphtheria toxin across the membrane of purified endosomes.

Translocation is a necessary and rate-limiting step for diphtheria toxin (DT) cytotoxicity. We have reconstituted DT translocation in a cell-free system using endosomes purified from lymphocytes and have demonstrated this using two different probe/cell systems, which provided identical results: 125I-DT/human CEM cells and 125I-transferrin-DT/mouse BW cells. The cell-free DT translocation process was found to be dependent on the presence of the pH gradient endosome (pH 5.3)/cytosol (pH 7). Among the pH equilibrating agents, nigericin (5 microM) was found to be the most effective, inhibiting DT translocation by 88%. An optimum pH value of 7 on the cytosolic side of the membrane (pH gradient approximately 1.7) was determined. ATP per se is not required for DT translocation. 125I-DT translocation was 3-fold more active from late than from early endosomes, probably because of their slightly more acidic pH. Only the A chain of the toxin was found to escape from either 125I-DT/CEM or 125I-transferrin-DT/BW endosomes. Translocation of control endosome labels (125I-transferrin and 125I-horseradish peroxidase) was never observed. We also show that DT receptors present on resistant (mouse) cells block the translocation of the toxin and are responsible for the resistance of these cells to DT.     

Effects of dynamin inactivation on pathways of anthrax toxin uptake

Internalization and traffic to acidic endosomes of anthrax lethal factor (LF) and protective antigen (PA), bound to the anthrax toxin receptor (ATR), is required for LF translocation into the cytosol, where it can elicit its toxic effects. Dynamin is required for clathrin-mediated endocytosis, and long-term disruption of dynamin function blocks internalization of PA. We have used LFn-DTA, a surrogate of LF consisting of the N-terminal domain of LF fused to the catalytic subunit of diphtheria toxin, to differentiate the effects of acute and long-term block of dynamin function on LFn-DTA toxicity. Both forms of interference reduce LFn-DTA toxicity only partially, consistent with alternative routes for LFn-DTA endocytosis. In contrast, a long-term block of dynamin activity results in a further interference with LFn-DTA toxicity that is consistent with an altered endosomal environment, probably an increase in endosomal pH.     

Activation of rat liver adenylate cyclase by cholera toxin requires toxin internalization and processing in endosomes

Involvement of acidic cell compartments in processing and action of cholera toxin (CT) in rat liver has been examined using subcellular fractionation. Liver cell fractions prepared various times after CT injection display, after a lag phase, a progressive increase in adenylate cyclase activity, detectable earlier in Golgi-endosomal fractions (20 min) than in plasma membrane fractions (30 min), with a maximum (3-fold basal activity) achieved by 60-90 min. Endosomes containing in vivo internalized CT display a time-dependent increase in their ability to bind anti-A-subunit antibodies and to stimulate exogenous adenylate cyclase, which kinetically parallels the generation of A1 peptide, suggesting a translocation of A-subunit (or A1 peptide) across the endosomal membrane. In vivo chloroquine treatment inhibits endocytosis of CT taken up into the liver, lengthens the lag phase for adenylate cyclase activation by CT, and reduces by 3- to 10-fold the apparent affinity of the toxin for the enzyme. Incubation of endosomes containing internalized toxin at 37 degrees C under isotonic conditions results in a pH-dependent increase in generation of A1 peptide, membrane translocation of A-subunit (or A1 peptide), and degradation of toxin, with a maximum at pH 5. Addition of ATP, by decreasing the internal endosomal pH, stimulates both generation of the A1 peptide and degradation of toxin at pH 6-8. It is concluded that activation of adenylate cyclase by CT in intact liver requires association and subsequent processing of toxin in an acidic cell compartment, presumably endosomal.     

Membrane translocation of diphtheria toxin fragment A exploits early to late endosome trafficking machinery

After reaching early endosomes by receptor-mediated endocytosis, diphtheria toxin (DT) molecules have two possible fates. A large pool enters the degradative pathway whereas a few molecules become cytotoxic by translocating their catalytic fragment A (DTA) into the cytosol. Impairment of DT degradation by microtubule depolymerization does not block DT cytotoxicity. Therefore, DTA membrane translocation into the cytosol occurs from an endocytic compartment located upstream of late endosomes. Comparisons between early endosomes and endocytic carrier vesicles in a cell-free translocation assay have demonstrated that early endosomes are the earliest endocytic compartment from which DTA translocates. DTA translocation is ATP-dependent, requires early endosomal acidification, and is increased by the addition of cytosol. Cytosol-dependent DTA translocation is GTPγS-insensitive but is blocked by anti-βCOP antibodies.     

Glucagon-mediated internalization of serine-phosphorylated glucagon receptor and Gsalpha in rat liver

To assess glucagon receptor compartmentalization and signal transduction in liver parenchyma, we have studied the functional relationship between glucagon receptor endocytosis, phosphorylation and coupling to the adenylate cyclase system. Following administration of a saturating dose of glucagon to rats, a rapid internalization of glucagon receptor was observed coincident with its serine phosphorylation both at the plasma membrane and within endosomes. Co-incident with glucagon receptor endocytosis, a massive internalization of both the 45- and 47-kDa Gsalpha proteins was also observed. In contrast, no change in the subcellular distribution of adenylate cyclase or beta-arrestin 1 and 2 was observed. In response to des-His(1)-[Glu(9)]glucagon amide, a glucagon receptor antagonist, the extent and rate of glucagon receptor endocytosis and Gsalpha shift were markedly reduced compared with wild-type glucagon. However, while the glucagon analog exhibited a wild-type affinity for endosomal acidic glucagonase activity and was processed at low pH with similar kinetics and rates, its proteolysis at neutral pH was 3-fold lower. In response to tetraiodoglucagon, a glucagon receptor agonist of enhanced biological potency, glucagon receptor endocytosis and Gsalpha shift were of higher magnitude and of longer duration, and a marked and prolonged activation of adenylate cyclase both at the plasma membrane and in endosomes was observed. The subsequent post-endosomal fate of internalized Gsalpha was evaluated in a cell-free rat liver endosome-lysosome fusion system following glucagon injection. A sustained endo-lysosomal transfer of the two 45- and 47-kDa Gsalpha isoforms was observed. Therefore, these results reveal that within hepatic target cells and consequent to glucagon-mediated internalization of the serine-phosphorylated glucagon receptor and the Gsalpha protein, extended signal transduction may occur in vivo at the locus of the endo-lysosomal apparatus.     

Interactions of cholera toxin with isolated hepatocytes. Effects of low pH, chloroquine and monensin on toxin internalization, processing and action.

The major steps in cholera-toxin action, i.e. binding, internalization, generation of A1 peptide and activation of adenylate cyclase, were examined in isolated hepatocytes. The binding of toxin involves a single class of high-affinity sites (KD congruent to 0.1 nM; Bmax. congruent to 10(7) sites/cell). At 37 degrees C, cell-associated toxin is progressively internalized, as judged by the loss of its accessibility to antibodies against whole toxin, A and B subunits (about 50, 75 and 30% of initially bound toxin after 40 min respectively). Two distinct pathways are involved in this process: endocytosis of the whole toxin, and selective penetration of the A subunit into the plasma membrane. Exposure of hepatocytes to an acidic medium (pH 5) results in a rapid and marked disappearance of the A subunit from the cell surface. Generation of A1 peptide and activation of adenylate cyclase by the toxin occur after a lag phase (10 min at 37 degrees C), and increase with time in a parallel manner up to 2-3% A1 peptide generated; they are unaffected by exposure of cells to an acidic medium. Chloroquine and monensin, which elevate the pH in acidic organelles, inhibit by 2-4-fold both the generation of A1 peptide and the activation of adenylate cyclase. Unexpectedly, these drugs also inhibit the internalization of the toxin. These results suggest that an acidic pH facilitates the penetration of A subunit into the plasma membrane and presumably the endosomal membrane as well, and that endocytosis of cholera toxin is required for generation of A1 peptide and activation of adenylate cyclase.     

Endosomes and toxin translocation

In this review we discuss data obtained by our group regarding the entry of toxins, especially ricin, diphtheria toxin (DT) and Pseudomonas exotoxin A (PE) into animal cells. We studied the translocation process of these toxins using endosomes purified from lymphocytes. This process is rate-limiting for toxicity and enables these toxins to reach the cytosol where they will inactivate the protein synthesis system and kill the cell. We could show that each of these toxins uses a different strategy to cross the endosome membrane. Whereas ricin transmembrane transport only relies on cytosolic ATP hydrolysis, PE first requires exposure to the low endosomal pH (pH-6), presumably to insert into the endosome membrane, before being translocated via a process which also requires cytosolic ATP hydrolysis. DT translocation is directly triggered and energized by the endosome-cytosol pH gradient. Using conjugates with dihydrofolate reductase we could indirectly show that ricin and PE require unfolding for translocation. A deletion approach enabled to produce a more cytotoxic PE mutant by increasing its translocation activity.     

Intracellular localization and degradation of diphtheria toxin

The internalization of surface-bound diphtheria toxin (DT) in BS-C-1 cells correlated with its appearance in intracellular endosomal vesicles; essentially no toxin appeared within secondary lysosomal vesicles. In contrast, internalized epidermal growth factor (EGF) was localized within both endosomal and lysosomal vesicles. Upon preincubation of cells with leupeptin, a lysosomal protease inhibitor, a threefold increase in the accumulation of EGF into lysosomes was observed. Under identical conditions, essentially all of the diphtheria toxin remained within endosomes (less than 2% of the intracellular diphtheria toxin accumulated in the lysosomal fraction), indicating that the inability to detect diphtheria toxin in lysosomes was not due to its rapid turnover within this vesicle. Following internalization of EGF or DT, up to 40% of the ligand appeared in the medium as TCA-soluble radioactivity. EGF degradation was partially leupeptin-sensitive and markedly NH4Cl-sensitive, indicating lysosomal degradation. In contrast, DT A-fragment degradation was resistant to these inhibitors, while B-fragment showed only partial sensitivity. These data suggest that the bulk of endocytosed diphtheria toxin is localized within endosomes and degraded by a pathway essentially independent of lysosomes.     

Involvement of ATP-dependent Pseudomonas Exotoxin Translocation from a Late Recycling Compartment in Lymphocyte Intoxication Procedure

Pseudomonas exotoxin (PE) is a cytotoxin which, after endocytosis, is delivered to the cytosol where it inactivates protein synthesis. Using diaminobenzidine cytochemistry, we found over 94% of internalized PE in transferrin (Tf) -positive endosomes of lymphocytes. When PE translocation was examined in a cell-free assay using purified endocytic vesicles, more than 40% of endosomal ¹²⁵I-labeled PE was transported after 2 h at 37°C, whereas a toxin inactivated by point mutation in its translocation domain was not translocated. Sorting of endosomes did not allow cell-free PE translocation, whereas active PE transmembrane transport was observed after > 10 min of endocytosis when PE and fluorescent-Tf were localized by confocal immunofluorescence microscopy within a rab5-positive and rab4- and rab7-negative recycling compartment in the pericentriolar region of the cell. Accordingly, when PE delivery to this structure was inhibited using a 20°C endocytosis temperature, subsequent translocation from purified endosomes was impaired. Translocation was also inhibited when endosomes were obtained from cells labeled with PE in the presence of brefeldin A, which caused fusion of translocation-competent recycling endosomes with translocation-incompetent sorting elements. No PE processing was observed in lymphocyte endosomes, the full-sized toxin was translocated and recovered in an enzymatically active form. ATP hydrolysis was found to directly provide the energy required for PE translocation. Inhibitors of endosome acidification (weak bases, protonophores, or bafilomycin A1) when added to the assay did not significantly affect ¹²⁵I-labeled PE translocation, demonstrating that this transport is independent of the endosome-cytosol pH gradient. Nevertheless, when ¹²⁵I-labeled PE endocytosis was performed in the presence of one of these molecules, translocation from endosomes was strongly inhibited, indicating that exposure to acidic pH is a prerequisite for PE membrane traversal. When applied during endocytosis, treatments that protect cells against PE intoxication (low temperatures, inhibitors of endosome acidification, and brefeldin A) impaired ¹²⁵I-labeled PE translocation from purified endosomes. We conclude that PE translocation from a late receptor recycling compartment is implicated in the lymphocyte intoxication procedure.     

The E5 oncoprotein of human papillomavirus type 16 inhibits the acidification of endosomes in human keratinocytes.

The human papillomavirus type 16 E5 oncoprotein possesses mitogenic activity that acts synergistically with epidermal growth factor (EGF) in human keratinocytes and inhibits the degradation of the EGF receptor in endosomal compartments after ligand-stimulated endocytosis. One potential explanation for these observations is that E5 inhibits the acidification of endosomes. This may be mediated through the 16-kDa component of the vacuolar proton-ATPase, since animal and human papillomavirus E5 proteins bind this subunit protein. Using a ratio-imaging technique to determine endosomal pH, we found that the acidification of endosomes in E5-expressing keratinocytes was delayed at least fourfold compared with normal human keratinocytes and endosomes in some cells never completely acidified. Furthermore, E5 expression increased the resistance of keratinocytes to protein synthesis inhibition by diphtheria toxin, a process dependent on efficient endosomal acidification. Finally, artificially inhibiting endosomal acidification with chloroquine during the endocytosis of EGF receptors in keratinocytes demonstrated many of the same effects as the expression of human papillomavirus type 16 E5, including prolonged retention of undegraded EGF receptors in intracellular vesicles.     

Roles of Glu 349 and Asp 352 in membrane insertion and translocation by diphtheria toxin.

Acidic conditions within the endosomal lumen induce the T domain of receptor-bound diphtheria toxin (DT) to insert into the endosomal membrane and mediate translocation of the toxin's catalytic domain to the cytosol. A conformational rearrangement in the toxin occurring near pH5 allows a buried apolar helical hairpin of the native T domain (helices TH8 and TH9) to undergo membrane insertion. If the inserted hairpin spans the bilayer, as hypothesized, then the two acidic residues within the TL5 interhelical loop, Glu 349 and Asp 352, should become exposed at the neutral cytosolic face of the membrane and reionize. To investigate the roles of these residues in toxin action, we characterized mutant toxins in which one or both acidic residues had been replaced with nonionizable ones. Each of two double mutants examined showed a several-fold reduction in cytotoxicity in 24-h Vero cell assays (sixfold for E349A + D352A and fourfold for E349Q + D352N), whereas the individual E349Q and D352N mutations caused smaller reductions in toxicity. The single and double mutations also attenuated the toxin's ability to permeabilize Vero cells to Rb+ at low pH and decreased channel formation by the toxin in artificial planar bilayers. Neither of the double mutations affected the pH-dependence profile of the toxin's conformational rearrangement in solution, as measured by binding of the hydrophobic fluorophore, 2-p-toluidinyl-naphthalene 6-sulfonate. The results demonstrate that, although there is no absolute requirement for an acidic residue within the TL5 loop for toxicity, Glu 349 and Asp 352 do significantly enhance the biological activity of the protein. The data are consistent with a model in which ionization of these residues at the cytosolic face of the endosomal membrane stabilizes the TH8/TH9 hairpin in a transmembrane configuration, thereby facilitating channel formation and translocation of the toxin's catalytic chain.     

IgG transcytosis and recycling by FcRn expressed in MDCK cells reveals ligand-induced redistribution

The neonatal Fc receptor (FcRn) transports IgG across epithelial cells and recycles serum IgG. FcRn binds IgG at the acidic pH of endosomes and releases IgG at the basic pH of blood. We expressed rat FcRn in polarized MDCK cells and demonstrated that it functions in transcytosis and recycling of IgG. In the absence of IgG, FcRn is distributed predominantly apically, but redistributes to basolateral locations upon IgG addition, indicating that ligand binding induces a signal that stimulates transcytosis. FcRn transcytoses IgG more efficiently in the apical to basolateral than the reverse direction when IgG is internalized by receptor-mediated endocytosis at acidic pH or by fluid phase endocytosis at basic pH. The PI 3-kinase inhibitor wortmannin disrupts basolateral recycling and transcytosis in both directions, but only minimally reduces apical recycling. Confocal imaging and quantitative IgG transport studies demonstrate that apically-internalized IgG recycles to the apical surface mainly from wortmannin-insensitive apical early endosomes, whereas FcRn-IgG complexes that transcytose to the basolateral surface pass through downstream Rab11-positive apical recycling endosomes and transferrin-positive common endosomal compartments.     

Oligomerization of membrane-bound diphtheria toxin (CRM197) facilitates a transition to the open form and deep insertion

Diphtheria toxin (DT) contains separate domains for receptor-specific binding, translocation, and enzymatic activity. After binding to cells, DT is taken up into endosome-like acidic compartments where the translocation domain inserts into the endosomal membrane and releases the catalytic domain into the cytosol. The process by which the catalytic domain is translocated across the endosomal membrane is known to involve pH-induced conformational changes; however, the molecular mechanisms are not yet understood, in large part due to the challenge of probing the conformation of the membrane-bound protein. In this work neutron reflection provided detailed conformational information for membrane-bound DT (CRM197) in situ. The data revealed that the bound toxin oligomerizes with increasing DT concentration and that the oligomeric form (and only the oligomeric form) undergoes a large extension into solution with decreasing pH that coincides with deep insertion of residues into the membrane. We interpret the large extension as a transition to the open form. These results thus indicate that as a function of bulk DT concentration, adsorbed DT passes from an inactive state with a monomeric dimension normal to the plane of the membrane to an active state with a dimeric dimension normal to the plane of the membrane.     

HIV-1 Tat enters T cells using coated pits before translocating from acidified endosomes and eliciting biological responses

The HIV-1 Tat protein is secreted by infected cells. Extracellular Tat can affect bystander uninfected T cells and induce numerous biological responses such as apoptosis and cytokine secretion. Tat is likely involved in several immune disorders during AIDS. Nevertheless, it is not known whether Tat triggers cell responses directly upon binding to signaling receptors at the plasma membrane or after delivery to the cytosol. The pathway that enables Tat to reach the cytosol is also unclear. Here we visualized Tat within T-cell-coated pits and endosomes. Moreover, inhibitors of clathrin/AP-2-mediated uptake such as chlorpromazine, activated RhoA, or dominant-negative mutants of Eps15, intersectin, dynamin, or rab5 impaired Tat delivery to the cytosol by preventing its endocytosis. Molecules neutralizing low endosomal pH or Hsp90 inhibitors abolished Tat entry at a later stage by blocking its endosomal translocation, as directly shown using a cell-free translocation assay. Finally, endosomal pH neutralization prevented Tat from inducing T-cell responses such as NF-kappaB activation, apoptosis, and interleukin secretion, indicating that cytosolic delivery is required for Tat signaling. Hence, Tat enters T cells essentially like diphtheria toxin, using clathrin-mediated endocytosis before low-pH-induced and Hsp90-assisted endosomal translocation. Cell responses are then induced from the cytosol.     

The host cell chaperone Hsp90 is essential for translocation of the binary Clostridium botulinum C2 toxin into the cytosol

Clostridium botulinum C2 toxin is the prototype of the binary actin-ADP-ribosylating toxins and consists of the binding component C2II and the enzyme component C2I. The activated binding component C2IIa forms heptamers, which bind to carbohydrates on the cell surface and interact with the enzyme component C2I. This toxin complex is taken up by receptor-mediated endocytosis. In acidic endosomes, heptameric C2IIa forms pores and mediates the translocation of C2I into the cytosol. We report that the heat shock protein (Hsp) 90-specific inhibitors, geldanamycin or radicicol, block intoxication of Vero cells, rat astrocytes, and HeLa cells by C2 toxin. ADP-ribosylation of actin in the cytosol of toxin-treated cells revealed that less active C2I was translocated into the cytosol after treatment with Hsp90 inhibitors. Under control conditions, C2I was localized in the cytosol of toxin-treated rat astrocytes, whereas geldanamycin blocked the cytosolic distribution of C2I. At low extracellular pH (pH 4.5), which allows the direct translocation of C2I via C2IIa heptamers across the cell membrane into the cytosol, Hsp90 inhibitors retarded intoxication by C2I. Geldanamycin did not affect toxin binding, endocytosis, and pore formation by C2IIa. The ADP-ribosyltransferase activity of C2I was not affected by Hsp90 inhibitors in vitro. The cytotoxic actions of the actin-ADP-ribosylating Clostridium perfringens iota toxin and the Rho-ADP-ribosylating C2-C3 fusion toxin was similarly blocked by Hsp90 inhibitors. In contrast, radicicol and geldanamycin had no effect on anthrax lethal toxin-induced cytotoxicity of J774-A1 macrophage-like cells or on cytotoxic effects of the glucosylating Clostridium difficile toxin B in Vero cells. The data indicate that Hsp90 is essential for the membrane translocation of ADP-ribosylating toxins delivered by C2II.