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.     

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.     

Diphtheria toxin entry into cells is facilitated by low pH

At neutral pH, NH4Cl and chloroquine protected cells against diphtheria toxin. A brief exposure of the cells to low pH (4.5-5.5) at 37 degrees completely abolished this protection. When, to cells preincubated with diphtheria toxin and NH4Cl, neutralizing amounts of anti-diphtheria toxin were added before the pH was lowered, the toxic effect was considerably reduced, but it was not completely abolished. A much stronger toxic effect was seen when antibodies were added immediately after incubation at low pH. Upon a short incubation with diphtheria toxin at low pH, the rate of protein synthesis in the cells decreased much faster than when the normal pH was maintained. The data suggest that, at low pH, diphtheria toxin (or its A fragment) penetrates directly through the surface membrane of the cell. The possibility is discussed that, when the medium has a neutral pH, the entry of diphtheria toxin involves adsorptive endocytosis and reduction of the pH in the vesicles possibly by fusion with lysosomes. Low pH did not facilitate the entry of the closely related toxins abrin, ricin, and modeccin.     

Evidence that diphtheria toxin and modeccin enter the cytosol from different vesicular compartments

Inhibition of protein synthesis in Vero cells was measured at different periods of time after treatment with diphtheria toxin and the related plant toxin modeccin. Diphtheria toxin acted much more rapidly than modeccin. Cells were protected against both toxins with antiserum as well as with agents like NH4Cl, procaine, and the ionophores monensin, FCCP, and CCCP, which increase the pH of intracellular vesicles. Antiserum, which is supposed to inactivate toxin only at the cell surface, protected only when it was added within a short period of time after modeccin. Compounds that increase the pH of intracellular vesicles, protected even when added after 2 h, indicating that modeccin remains inside vesicles for a considerable period of time before it enters the cytosol. After addition of diphtheria toxin to the cells, compounds that increase the pH of intracellular vesicles protected only approximately to the same extent as antitoxin. This indicates that after endocytosis diphtheria toxin rapidly enters the cytosol. At 20 degrees C, the cells were more strongly protected against modeccin than against diphtheria toxin. The residual toxic effect of diphtheria toxin at 20 degrees C could be blocked with NH4Cl whereas this was not the case with modeccin. This indicates that at 20 degrees C the uptake of diphtheria toxin occurs by the normal route, whereas the uptake of modeccin occurs by a less efficient route than that dominating at 37 degrees C. The results indicate that after endocytosis diphtheria toxin rapidly enters the cytosol from early endosomes with low pH (receptosomes). Modeccin enters the cytosol much more slowly, possibly after fusion of the endocytic vesicles with another compartment.     

pH-dependent bilayer destabilization and fusion of phospholipidic large unilamellar vesicles induced by diphtheria toxin and its fragments A and B

The passage by the low endosomal pH is believed to be an essential step of the diphtheria toxin (DT) intoxication process in vivo. Several studies have suggested that this low pH triggers the insertion of DT into the membrane. We demonstrate here that its insertion into large unilamellar vesicles (LUV) is accompanied by a strong destabilization of the vesicles at low pH. The destabilization has been studied by following the release of a fluorescent dye (calcein) encapsulated in the liposomes. The influence of the lipid composition upon this process has been examined. At a given pH, the calcein release is always faster for a negatively charged (asolectin) than for a zwitterionic (egg PC) system. Moreover, the transition pH, which is the pH at which the toxin-induced release becomes significant, is shifted upward for the asolectin LUV as compared to the egg PC LUV. No calcein release is observed for rigid phospholipid vesicles (DPPC and DPPC/DPPA 9/1 mol/mol) below their transition temperature whereas DT induces an important release of the dye in the temperature range corresponding to the phase transition. The transition pH associated to the calcein release from egg PC vesicles is identical with that corresponding to the exposure of the DT hydrophobic domains, as revealed here by the binding of a hydrophobic probe (ANS) to the toxin. This suggests the involvement of these domains in the destabilization process. Both A and B fragments destabilize asolectin and PC vesicles in a pH-dependent manner but to a lesser extent than the entire toxin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endosomal proteolysis of diphtheria toxin without toxin translocation into the cytosol of rat liver in vivo

A detailed proteolysis study of internalized diphtheria toxin (DT) within rat liver endosomes was undertaken to determine whether DT-resistant species exhibit defects in toxin endocytosis, toxin activation by cellular enzymes or toxin translocation to its cytosolic target. Following administration of a saturating dose of wild-type DT or nontoxic mutant DT (mDT) to rats, rapid endocytosis of the intact 62-kDa toxin was observed coincident with the endosomal association of DT-A (low association) and DT-B (high association) subunits. Assessment of the subsequent post-endosomal fate of internalized mDT revealed a sustained endo-lysosomal transfer of the mDT-B subunit accompanied by a net decrease in intact mDT and mDT-A subunit throughout the endo-lysosomal apparatus. In vitro proteolysis of DT, using an endosomal lysate, was observed at both neutral and acidic pH, with the subsequent generation of DT-A and DT-B subunits (pH 7) or DT fragments with low ADP-ribosyltransferase activity (pH 4). Biochemical characterization revealed that the neutral endosomal DT-degrading activity was due to a novel luminal 70-kDa furin enzyme, whereas the aspartic acid protease cathepsin D (EC 3.4.23.5) was identified as being responsible for toxin degradation at acidic pH. Moreover, an absence of in vivo association of the DT-A subunit with cytosolic fractions was identified, as well as an absence of in vitro translocation of the DT-A subunit from cell-free endosomes into the external milieu. Based on these findings, we propose that, in rat, resistance to DT may originate from two different mechanisms: the ability of free DT-A subunits to be rapidly proteolyzed by acidic cathepsin D within the endosomal lumen, and/or the absence of DT translocation across the endosomal membrane, which may arise from the absence of a functional cytosolic translocation factor previously reported to participate in the export of DT from human endosomes.     

Requirements for the translocation of diphtheria toxin fragment A across lipid membranes

The translocation of the enzymatic moiety of diphtheria toxin, fragment A, across the membranes of pure lipid vesicles was demonstrated. A new assay, which employed vesicles made to contain radiolabeled NAD and elongation factor-2, was used to measure the appearance of the enzymatic activity of the A fragment in the vesicles. When the vesicles were exposed to a low-pH medium in the presence of diphtheria toxin, small molecules, such as NAD, escaped into the extravesicular medium, whereas large molecules mostly remained inside the vesicles. The vesicle-entrapped elongation factor-2 became ADP-ribosylated, indicating the entry of fragment A into the vesicle. The translocation of the A fragment depended upon the pH of the medium, being negligible at pH greater than 7.0 and maximal at pH 4.5. The entire toxin molecule was needed for function; neither the A fragment nor the B fragment alone was able to translocate itself across and react with the sequestered substrates. After exposure of the toxin to low pH, the entry of the A fragment was rapid, being virtually complete within 2-3 min at pH 5.5, and within 1 min at pH 4.7. Translocation occurred in the absence of any protein in the vesicle membrane. These results are consistent with the notion that the diphtheria toxin molecule enters the cytoplasm of a cell by escaping from an acidic compartment such as an endocytic vesicle.     

Reversible refolding of the diphtheria toxin T-domain on lipid membranes

The catalytic domain of diphtheria toxin (DT) is translocated across endosomal membranes by the T-domain (DTT) in response to acidification. Understanding the energetics of translocation, besides clarifying the mechanism of translocation, should provide insights into general principles of membrane protein stability and assembly. As a first step, we have evaluated the energetics of DTT binding to lipid vesicles using three single-cysteine mutants (L350C, Q369C, and Y280C) labeled with a 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) fluorophore sensitive to polarity changes. Remarkably strong association with the vesicles was detected for all mutants, even at pH 7 at which DTT is believed to be in a fully folded membrane-incompetent state. Lowering the pH in the presence of anionic membranes resulted in a strong but reversible increase in emission of NBD-labeled mutants, consistent with reversible membrane insertion. This reversibility permitted free energies of DTT interactions with vesicles to be determined for the first time. Free energy values for the three mutants ranged from -8 to -10 kcal mol(-1) at pH 4.3 and from -7 to -8 kcal mol(-1) at pH 7. Insights into the disposition of DTT on membranes were obtained using a novel hydropathy analysis that considers the relative free energies of transmembrane and interfacial interactions as a function of pH. This analysis suggests that interactions at the membrane interface dominate pH-triggered insertion of DTT, implying that the folding pathway involves interfacial intermediates.     

Characterization of diphtheria toxin-induced lesions in liposomal membranes. An evaluation of the relationship between toxin insertion and "channel" formation

Diphtheria toxin interaction with membranes has been studied by following the release of a fluorescent dye (calcein) encapsulated within large unilamellar vesicles. Results showed that diphtheria toxin induced temperature- as well as pH-dependent permeability changes in these model membranes. Interestingly, insertion of the "channel-forming" B domain was not sufficient for calcein release, since dye release from vesicles composed of dimyristoyllecithin:cholesterol:dicetylphosphate 4:3:0.8) was completely inhibited at low temperatures which permitted B insertion. Rather, the temperature dependence of calcein release from and A domain insertion into dimyristoyllecithin:cholesterol:dicetyl phosphate vesicles suggest some relationship between "channel formation" and fragment A translocation across membranes. However, the nature of the toxin channel is called into question by our observations that channel size, in addition to activity, was pH-dependent. On the basis of these experiments, it is proposed that the toxin "channel" is the result of localized perturbations in the lipid bilayer at the interface between lipids and inserted toxin molecules that are sufficiently large in fluid membranes at low pH to allow the translocation of fragment A across the bilayer.     

Monensin blocks the transport of diphtheria toxin to the cell cytoplasm

Lysosomotropic amines are believed to inhibit the transport of diphtheria toxin to the cell cytoplasm by raising the pH within intracellular vesicles. If so, then other drugs that dissipate intracellular proton gradients should have a similar effect on toxin transport. We found that monensin, a proton ionophore unrelated to lysosomotropic amines, is a potent inhibitor of the cytotoxic effect of diphtheria toxin. Monensin appears to block the escape of endocytosed toxin from a vesicle to the cytoplasm. Monensin fails to protect cells from the effects of diphtheria toxin that is bound to the cell surface and exposed to acidic medium, suggesting that the step normally blocked by the drug is circumvented under these conditions. The inhibition of toxin transport caused by monensin could not be relieved when monensin was replaced by ammonium chloride, nor when ammonium chloride was again replaced by monensin. This suggests that both drugs block the same step of toxin transport. The effect of monensin on the transport of diphtheria toxin to the cytoplasm is consistent with the proposal (Draper and Simon. 1980. J. Cell Biol. 87:849-854; Sandvig and Olsnes. 1980. J. Cell Biol. 87:828-832) that the toxin is endocytosed and then, in response to an acidic environment, penetrates through the membrane of an intracellular vesicle to reach the cytoplasm.     

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.     

Topology of diphtheria toxin B fragment inserted in lipid vesicies

Diphtheria toxin (DT) is a bacterial protein that crosses the membrane of endosomes of target cells In response to the low endosomal pH. In this paper, we have inserted diphtheria toxin in asolectin vesicles at pH 5.0 and treated the reconstituted system with pronase. The peptides that were protected from digestion were separated by gel electrophoresls, transferred to a membrane and their N-terminal sequences were determined. All peptides belong to the B fragment of DT and cover residues 194–223, 266–375 and 429–528. The secondary structures of the peptides inserted in the membrane, determined by Fourier-transformed infrared spectroscopy, were shown to be mostly α-helices and β-sheets (44% and 53%, respectively). On the basis of these data and the recently published X-ray structure of DT, we are proposing a topology for the DTB fragment in the membrane.     

Lipid interaction of diphtheria toxin and mutants with altered fragment B. 1. Liposome aggregation and fusion

The interaction of diphtheria toxin and its cross-reacting mutants crm 45,228 and 1001 with small unilamellar vesicles has been followed by a turbidity assay, electron microscopy, fluorescence energy transfer and membrane permeability. All toxins at pH lower than 6 induce the aggregation and fusion of liposomes containing negatively charged phospholipids; crm 45 and crm 1001 are less potent than diphtheria toxin. Isolated diphtheria toxin fragment B is very effective while isolated fragment A is ineffective. Liposome fusion induced by the toxins at low pH occurs without release of the internal content implying that fusion does not involve vesicle breakage and resealing. The pH dependence of the membrane interaction of diphtheria toxin monitored by turbidity is in close agreement with that monitored by fluorescence energy transfer. It shows that diphtheria toxin can alter the lipid bilayer structure in the pH interval 5-6. This pH range occurs in endosomes and suggests that histidyl and carboxyl residues are likely to be involved in the conformational change of diphtheria toxin triggered by acidic pH.     

Interaction of diphtheria toxin with model membranes

Low pH is believed to trigger membrane penetration by diphtheria toxin in vivo. The effect of pH upon the binding of the toxin to unilamellar model membrane vesicles was determined by using a fluorescence quenching assay. A series of studies were undertaken to determine the effect of lipid composition upon the binding of lipids to the toxin. The binding of toxin to various small unilamellar vesicles of zwitterionic or anionic lipids was similar in extent and was accompanied by deep penetration of the toxin into the fatty acyl chains, in agreement with previous studies. However, the transition pH, which is the pH at and below which toxin binding becomes significant, depended upon the fraction of anionic lipids, being highest with model membranes composed totally of anionic lipids (pH 5.8) and lowest with membranes composed of zwitterionic lipids (pH 5.2). Except for vesicle charge, the transition pH was independent of the nature of the lipid polar groups used. High ionic strength, which had no effect on the transition pH with zwitterionic vesicles, was found to shift the transition pH with totally anionic vesicles to pH 5.2. This suggests that both direct protein-lipid electrostatic interactions and the ionic double layer, which gives rise to a low local pH around anionic vesicles, contribute to the shift in the transition pH. The effect of lipid composition upon the kinetics and strength of binding was also examined. At low pH, binding was rapid and tight. Binding to vesicles containing 20 wt % anionic phosphatidylglycerol was faster and tighter than binding to vesicles of zwitterionic phosphatidylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)     

The pH-dependent conformational change of diphtheria toxin

Labeling by a hydrophobic photoactivatable reagent and limited proteolysis have been used to study conformational changes of diphtheria toxin related to its pH-dependent membrane insertion and translocation. TID (3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine) labels diphtheria toxin at pH 5 much more efficiently than at pH 7, both in the presence and absence of lipid vesicles. In the absence of membranes, the extent of labeling is greater and the pH dependence is stronger. As analyzed on sodium dodecyl sulfate-polyacrylamide gels and by high pressure liquid chromatography, both the A- and B-subunits and most of the cyanogen bromide fragments of the toxin are labeled by TID at acid pH. The products of trypsin cleavage of diphtheria toxin at pH 5 are different from those seen at neutral pH. Trypsin-susceptible sites were identified by gel electrophoresis of the trypsin fragments, combined with electrophoresis and high pressure liquid chromatography of CNBr digests of trypsin-treated toxin. At neutral pH, the main sites of digestion are at the junction between the A- and B-fragments and near the NH2 terminus of the A-fragment. At pH 5.2, these sites are less efficiently cut, and new sites appear near the NH2 terminus of the B-fragment, in an amphipathic portion of the sequence. Thus, even in the absence of membranes, acid pH induces a significant conformational change in diphtheria toxin. This change involves burial of some previously accessible sites, exposure of previously inaccessible sites, and the formation of hydrophobic regions over an extensive portion of the polypeptide chain.     

Interactions of diphtheria toxin with lipid vesicles: determinants of ion channel formation

Lipid vesicles have been utilized to study the interactions of diphtheria toxin (DT) with membranes. The assay for DT ion channel formation was fluorescence-detected membrane potential depolarization of vesicles in which valinomycin-induced potassium diffusion gradients had been generated. The following requirements for ion channel formation have been identified: (1) acid pH (less than 5); (2) trans-negative membrane potentials (35-fold increase in channel-forming activity from -6 mV to -59 mV); and (3) negatively charged phospholipid headgroups (about 100-fold more activity using vesicles formed from asolectin compared to soybean phosphatidylcholine). Concentration dependence plots of toxin activity showed a linear response with logarithmic slopes of nearly one for each lipid composition. These results show a close parallel to those obtained previously with planar lipid bilayers and thus provide guidelines for conditions which facilitate functional insertion of the toxin into vesicles.     

GRP78(BiP) facilitates the cytosolic delivery of anthrax lethal factor (LF) in vivo and functions as an unfoldase in vitro

Anthrax toxin is an A/B bacterial protein toxin which is composed of the enzymatically active Lethal Factor (LF) and/or Oedema Factor (EF) bound to Protective Antigen 63 (PA63) which functions as both the receptor binding and transmembrane domains. Once the toxin binds to its cell surface receptors it is internalized into the cell and traffics through Rab5- and Rab7-associated endosomal vesicles. Following acidification of the vesicle lumen, PA63 undergoes a dynamic change forming a beta-barrel that inserts into and forms a pore through the endosomal membrane. It is widely recognized that LF, and the related fusion protein LFnDTA, must be completely denatured in order to transit through the PA63 formed pore and enter the eukaryotic cell cytosol. We demonstrate by protease protection assays that the molecular chaperone GRP78 mediates the unfolding of LFnDTA and LF at neutral pH and thereby converts these proteins from a trypsin resistant to sensitive conformation. We have used immunoelectron microscopy and gold-labelled antibodies to demonstrate that both GRP78 and GRP94 chaperones are present in the lumen of endosomal vesicles. Finally, we have used siRNA to demonstrate that knock-down of GRP78 results in the emergence of resistance to anthrax lethal toxin and oedema toxin action.     

Diphtheria Toxin Forms Pores of Different Sizes Depending on Its Concentration in Membranes: Probable Relationship to Oligomerization

Diphtheria toxin forms pores in biological and model membranes upon exposure to low pH. These pores may play a critical role in the translocation of the A chain of the toxin into the cytoplasm. The effect of protein concentration on diphtheria toxin pore formation in model membrane systems was assayed by using a new fluorescence quenching method. In this method, the movement of Cascade Blue labeled dextrans of various sizes across membranes is detected by antibodies which quench Cascade Blue fluorescence. It was found that at low pH the toxin makes pores in phosphatidylcholine/phosphatidylglycerol vesicles with a size that depends on protein concentration. At the lowest toxin concentrations only the entrapped free fluorophore (MW 538) could be released from model membranes. At intermediate toxin concentrations, a 3 kD dextran could be released. At the highest toxin concentration, a 10 kD dextran could be released, but not a 70 kD dextran. Similar pore properties were found using vesicles lacking phosphatidylglycerol or containing 30% cholesterol. However, larger pores formed at lower protein concentrations in the presence of cholesterol. The dependence of pore size on toxin concentration suggests that toxin oligomerization regulates pore size. This behavior may explain some of the conflicting data on the size of the pores formed by diphtheria toxin. The formation of oligomers by membrane-inserted toxin is consistent with the results of chemical crosslinking and measurements of the self-quenching of rhodamine-labeled toxin. Based on these experiments we propose diphtheria toxin forms oligomers with a variable stoichiometry, and that pore size depends on the oligomerization state. Reasons why oligomerization could assist proper membrane insertion of the toxin and other proteins that convert from soluble to membrane-inserted states are discussed. Received: 10 March 1999/Revised: 22 June 1999     

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.     

Characterization of a transferrin-diphtheria toxin conjugate

We report here the synthesis and properties of a hybrid toxin prepared by covalently coupling diphtheria toxin to transferrin. The purified material contained two major hybrid protein species and was highly cytotoxic to mouse LMTK- cells in culture, reducing protein synthesis by 50% in 24 h at a concentration of 1 ng/ml. Cytotoxic activity was completely abolished in the presence of exogenous transferrin or anti-transferrin or anti-diphtheria toxin, thus demonstrating that the hybrid toxin was intoxicating cells via their transferrin receptors and that both the diphtheria toxin and transferrin components of the conjugate were necessary for activity. NH4Cl, a drug that elevates the pH within acidic intracellular vesicles, also blocked cytotoxic activity, suggesting that a low intravesicular pH was required for activity. The inhibitory effect of NH4Cl could be abolished by exposing toxin-treated cells to acidic culture medium, further implicating an acid-dependent step in the mechanism of the hybrid toxin action. Studies on the kinetics of intoxication also implied that endocytosis and exposure to a low pH within vesicles were necessary for cytotoxicity. Altogether, the results suggest that the transferrin-diphtheria toxin conjugate binds to transferrin receptors and is internalized into acidic endocytic vesicles. The enzymatic moiety of diphtheria toxin then apparently enters the cytosol in response to the low pH and subsequently arrests protein synthesis.