Diphtheria Toxin Translocation across Endosome Membranes
A NOVEL CELL PERMEABILIZATION ASSAY REVEALS NEW DIPHTHERIA TOXIN FRAGMENTS IN ENDOCYTIC VESICLES*

Toshiyuki UmataDagger and Eisuke MekadaDagger §

From the Dagger  Division of Cell Biology, Institute of Life Science, and the § Cellular and Developmental Biology Division, Research Center for Innovative Cancer Therapy, Kurume University, Aikawa, Kurume, Fukuoka 839, Japan

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

By using cells overexpressing diphtheria toxin (DT) receptor and a novel method of permeabilizing the plasma membrane with a bacterial pore-forming toxin, specific translocation of fragment A to the cytosol was observed, whereas full-size DT and other minor species of DT-derived fragments were left in intracellular vesicles. The translocation competence of DT proteins with mutations in the transmembrane domain is consistent with their cytotoxicities. The charge-reversal mutants E349K and D352K do not translocate their fragment A to the cytosol, whereas D352N is partially competent. ADP-ribosyltransferase activity of fragment A is not required for translocation. Novel fragments of DT with apparent molecular masses of 28 and 35 kDa were detected in endocytic vesicles. The 28-kDa fragment consists of fragment A and an N-terminal piece of fragment B, whereas the 35-kDa fragment contains part of fragment B and may be complementary to the 28-kDa fragment. Time course studies show that the 28-kDa fragment appears in endocytic vesicles prior to translocation of fragment A to the cytosol, raising the possibility that the 28-kDa fragment is an intermediate in translocation. We present a model for translocation of fragment A that incorporates the observations made using the novel permeabilization method.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Translocation of proteins across membranes is an important process in living cells. In eukaryotic cells proteins located in plasma membrane, lysosomes, and the Golgi complex are all synthesized on ribosomes attached to the endoplasmic reticulum, co-translationally translocated into the lumen of endoplasmic reticulum, and then transferred to appropriate destinations. Secreted proteins are also translocated into the endoplasmic reticulum, followed by exocytosis. Proteins localized in mitochondria, peroxisomes, chloroplasts, and nuclei are synthesized in the cytosol and translocated post-translationally into those organelles. The mechanisms of membrane penetration by these newly synthesized proteins and the machinery needed for their translocation have been studied in great detail.

Translocation of proteins from outside the cell to inside also occurs. A number of protein toxins exert their toxic effects in mammalian cells by enzymatically modifying an intracellular target. At least a portion of the protein must be therefore be translocated across the cell membrane. Cholera toxin, Escherichia coli heat-labile toxin, and pertussis toxin ADP-ribosylate subunits of trimeric G proteins and cause constitutive activation of adenylate cyclases (1-3). Diphtheria toxin (DT)1 and Pseudomonas aeruginosa exotoxin A inactivate elongation factor 2 and inhibit protein synthesis (4, 5). Several plant toxins as well as the Shiga toxin inactivate ribosomes by hydrolyzing specific sites in ribosome RNA (6-8). Tetanus and botulinum neurotoxins cleave proteins that are part of the machinery for exocytosis in neurons (9). Other toxins, including Clostridium difficile toxin (10) and anthrax toxin (11), also affect intracellular targets.

The mechanisms by which the toxins penetrate membranes are not as well understood as those for newly synthesized cellular proteins. Further studies could increase our understanding of the general mechanism of membrane penetration by proteins and of the pathogenesis of microbial infections.

Among these toxins, DT is one of the most extensively studied with regard to the mechanism of translocation. Knowledge of its crystal structure (12) and the availability of detailed information about the DT receptor (13-16) offer distinct advantages for the study of membrane translocation. DT is a single polypeptide with a molecular weight of 58,342 (17). DT is proteolytically cleaved to two distinct fragments, fragments A and B, in the presence of reducing agents (18, 19). Fragment A (Mr = 21,145) contains the catalytic domain of the toxin, which inhibits protein synthesis by ADP-ribosylation of elongation factor 2 (4). Fragment B (Mr = 37,240) is responsible for binding to the receptor and for mediating the translocation of fragment A into cytoplasm (20). Analysis of the crystal structure of DT revealed that fragment B consists of two structurally and functionally separable domains, referred to as T (transmembrane) and R (receptor-binding) (12). The T domain consists of nine alpha -helices, whereas the R domain is a beta -barrel structure. Translocation of fragment A of DT to the cytosol is initiated by binding of DT to a specific receptor on the cell surface (21, 22), which is identical to the membrane-anchored form of heparin-binding EGF-like growth factor (pro-HB-EGF) (15).

DT is internalized into endocytic vesicles by a receptor-mediated process (23, 24). Several lines of evidence indicate that the acidic environment of the endocytic vesicle is required for the translocation of fragment A from endosome to cytosol (25-29). A conformational change of the toxin molecule takes place in the acidic vesicles, resulting in the insertion of the T domain into the endocytic vesicle membrane, and finally the enzymatically active fragment A is translocated from the endocytic vesicle to the cytosol, where it exerts its toxicity. Proteolytic cleavage ("nicking") between fragments A and B of the toxin and reduction of the interfragment disulfide bond are required for intoxication (30, 31). The membrane-bound protease furin (and probably other cellular proteases) can cleave intact DT after DT binds to the receptor (32). Reduction of the disulfide bond between the two fragments takes place either prior to translocation of fragment A from endosomes or upon entry of fragment A into the cytosol.

The insertion of fragment A into the membrane and its translocation to the cytosol have been studied by several methods. By using whole cells exposed to low pH after DT binding at 4 °C, mimicking the endosome environment (33, 34), the toxin can be translocated directly from the cell surface to the cytosol when cells are incubated at 37 °C. DT-induced pore formation has also been studied by measuring ion conductance or the release of radioactive ions from cells (35-37). By using these systems, the T domain of fragment B was shown to be important for insertion and translocation of fragment A. Furthermore, in combination with use of mutant forms of DT and knowledge of its crystal structure, a precise evaluation of the role of each helix of the T domain has been made (38). The use of these systems nevertheless raises several criticisms. The most fundamental criticism is that the model membrane or plasma membrane is clearly different from the endosomal membrane through which DT translocation naturally occurs. Thus, a system is required to measure the translocation of fragment A from endosome to cytosol.

We developed a method for observing translocation of DT fragment A from endosome to cytosol based on selective permeabilization of the plasma membrane with Clostridium perfringens theta -toxin. By using this method we studied the structural requirements for DT translocation and found that novel fragments of DT appear in endocytic vesicles during translocation.

    EXPERIMENTAL PROCEDURES
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Procedures
Results
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References

Materials-- DT and nicked DT were prepared as described previously (39). DT mutants E148S, E349K, D352N, and D352K (38, 40) were kindly provided by Drs. J. A. Silverman and R. J. Collier (Harvard Medical School). C. perfringens theta -toxin was the gift of Dr. Y. Ohno-Iwashita (Tokyo Metropolitan Institute of Gerontology). Murine EGF was purchased from Nakarai tesque (Kyoto, Japan). Protein G was purchased from Pierce. Calf serum was purchased from Sanko Junyaku Co., Ltd. (Tokyo, Japan). Pronase was purchased from Calbiochem. Bafilomycin A1 was purchased from Wako Pure Chem., Ltd. (Osaka, Japan).

Cells-- Vero-H cells, Vero cells overexpressing DT receptor/pro-HB-EGF (41), were grown in Eagle's minimum essential medium supplemented with nonessential amino acids, 100 units/ml penicillin G, 100 mg/ml streptomycin, and 10% calf serum. Cells were seeded into tissue culture dishes and incubated for 15 h before use in translocation experiments.

Antibodies-- Anti-DT polyclonal antibody was produced in our laboratory by immunizing rabbits with a diphtheria toxoid. Anti-murine EGF rabbit antiserum was purchased from Nakarai tesque (Kyoto, Japan).

Translocation Assay-- DT was labeled with Na125I using Enzymobeads (Bio-Rad) as reported previously (39). The labeled toxin had a specific activity of 2-3 × 107 cpm/µg. Vero-H cells were incubated with 100 ng/ml 125I-DT in HMEM (HEPES-buffered minimum Eagle's medium containing nonessential amino acids, pH adjusted to 7.2 unless otherwise stated) supplemented with 10% calf serum at 4 °C for 3 h. Unbound 125I-DT was removed with chilled washing buffer (150 mM NaCl, 20 mM HEPES, 10 mM KCl, 0.2 mM CaCl2, 0.2 mM MgCl2, pH 7.0) and then the cells were incubated in pre-warmed HMEM supplemented with 10% calf serum at 37 °C for 1 h. DT remaining on the cell surface was digested by incubation with pre-warmed Pronase solution (5 mg/ml in HMEM, pH 7.2) at 37 °C for 5 min. After the addition of chilled calf serum to the plate, cells were harvested to a tube, washed once with chilled HMEM supplemented with 10% calf serum, and then washed twice with chilled HMEM supplemented with 1 mg/ml fatty acid-free bovine serum albumin. The cells were suspended in chilled HMEM supplemented with 1 mg/ml fatty acid-free bovine serum albumin. Ninety µl of cell suspension was mixed with 10 µl of 10,000 units/ml theta -toxin and then allowed to stand in ice for 10 min. Nine hundred µl of cold HMEM containing 3.3% calf serum were added to neutralize membrane-unbound theta -toxin, and then the cells were incubated at 37 °C for 5 min to permeabilize the plasma membrane. Samples were centrifuged, and the supernatants and cell pellets were analyzed by immunoprecipitation and SDS-PAGE. For assays with EGF, experiments were performed in a similar manner using Vero cells instead of Vero-H cells.

Construction of Hybrid Toxin with Labeled Fragment A and Unlabeled Fragment B-- Hybrid toxin was constructed as described previously (42). Fragment A was labeled with Na125I. 125I-fragment A (16.8 µg) and unlabeled nicked DT (100 µg) were mixed in a final volume of 380 µl, and 20 µl of 0.5 M dithiothreitol was added to the mixture. The mixture was allowed to stand for 30 min at room temperature and dialyzed overnight against 100 ml of 10 mM sodium phosphate buffer, pH 7.2, without agitation at 4 °C. Then the mixture was dialyzed against 300 ml of the same buffer containing 50 mM o-phenanthroline and 10 mM CuSO4 for 6 h with two changes of buffer, followed by further dialysis against 10 mM sodium phosphate buffer, pH 7.2, for about 20 h to remove o-phenanthroline and CuSO4.

SDS-PAGE and Autoradiography-- After the translocation assay, the cytosol fraction, the remnant fraction or whole cell fraction was mixed with lysis buffer (10 mM HEPES, 0.3 M NaCl, 60 mM octylglucoside, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 10 mM iodoacetamide, 10 mM N-ethylmaleimide, 10 mg/ml chymostatin, 20 mg/ml antipain, pH 7.0). The lysate was incubated with anti-DT antibody conjugated to CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech) at 4 °C. SDS-PAGE sample buffer was added to the gel, and gel-associated proteins were analyzed by SDS-PAGE in the presence or absence of dithiothreitol. Labeled bands were detected with an imaging analyzer (BAS 2000, Fuji Co., Ltd.). For the samples containing EGF, rabbit anti-EGF antiserum and protein G-Sepharose (Amersham Pharmacia Biotech) were used for the immunoprecipitation.

Digestion of Cytosol by Pronase in Permeabilized Cells-- Permeabilized cells were treated with pre-warmed Pronase solution (10 mg/ml) at 37 °C for 10 min in the presence or absence of 1% Triton X-100. Phenylmethylsulfonyl fluoride (0.1 M) was added to the cell suspension, and the cells were centrifuged. The precipitate was lysed with lysis buffer, and the lysate was analyzed by immunoprecipitation and SDS-PAGE.

Assay for DT Cytotoxicity-- DT cytotoxicity was measured by assaying the rate of protein synthesis as described previously (43). Briefly, cells in a 24-well tissue culture plate were washed twice with ice-cold phosphate-buffered saline (150 mM NaCl, 2.7 mM KCl, 10 mM phosphate buffer, pH 7.2), and then 0.5 ml of HEPES-buffered Ham's F-12 medium, pH 7.2, supplemented with 10% calf serum was added. Various concentrations of DT were added to the culture medium, and the cells were incubated at 37 °C for 4 h followed by further incubation with 0.5 mCi/ml [3H]leucine (ICN) for 1 h. The radioactivity incorporated into protein was measured. The rate of protein synthesis in each culture was expressed as a percentage of the value obtained in control cultures without DT.

Subcellular Fractionation-- Subcellular fractionation was performed as described previously (29).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Translocation Assay Based on Selective Permeabilization of the Plasma Membrane-- Selective permeabilization of the plasma membrane using C. perfringens theta -toxin under conditions that leave the endosomal membranes intact can be used to follow translocation of DT fragment A from endosomes to the cytosol, as outlined in Fig. 1. Vero-H cells, stable transformants of Vero cells overexpressing human DTR/pro-HB-EGF (41), were incubated with 125I-labeled DT at 4 °C. The cells were washed to remove unbound protein and then incubated at 37 °C to allow internalization of the bound toxin. Labeled toxin remaining on the cell surface was removed by treatment with Pronase. The cells were treated with theta -toxin, which, like streptolysin O, binds to membrane cholesterol and makes pores, allowing cytosolic proteins to diffuse out of the cells (44, 45). Distribution of the labeled internalized protein between the cytosol and endosomes was then determined by centrifugation to produce a cytosol fraction (supernatant) and remnant fraction containing the endosomes (pellet). These fractions were analyzed by immunoprecipitation, SDS-PAGE, and autoradiography.


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Fig. 1.   Outline of the translocation assay. Vero-H cells were incubated with 125I-labeled DT at 4 °C for 3 h. Cells were washed to remove the unbound DT and incubated at 37 °C for an appropriate time to allow internalization and translocation of DT. The DT remaining on the cell surface was removed by treatment with Pronase, and then the cells were permeabilized with C. perfringens theta -toxin. Cell samples were centrifuged to provide a cytosol fraction (supernatant) and remnant fraction (pellet). For some experiments, Pronase-treated cells were also used without permeabilization as the whole cell fraction.

To monitor leakage of internalized material from endosomes a similar assay was carried out using 125I-EGF, because EGF is internalized into endosomes and delivered to lysosomes but is not translocated to the cytosol (46, 47). Vero cells were used rather than Vero-H cells, because EGF receptors on Vero-H cells are down-regulated by secreted HB-EGF.

In the whole cell fraction, comprised of Pronase-treated cells that were not permeabilized with theta -toxin, the material derived from 125I-labeled DT appears in bands corresponding to full-size DT, fragment A, a 28-kDa band, and a number of other minor bands (Fig. 2, lane 4). Very little labeled material is present in the position corresponding to fragment B. All the bands observed are derived from intracellular material, because DT bound to the cell surface is completely removed by Pronase under the conditions used (data not shown). Only a trace amount of fragment A or EGF is present in the supernatant from cells that were not treated with theta -toxin, 1-3% of the amount in the whole cell fraction (Fig. 2, lane 1).


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Fig. 2.   Selective permeabilization of the plasma membrane. A, Vero-H cells with receptor-bound 125I-labeled intact DT were incubated at 37 °C for 1 h. After treatment with Pronase, cells were incubated with 1000 units/ml of theta -toxin at 4 °C for 10 min and then incubated at 37 °C for 5 min with or without 3% calf serum. The cytosol fraction was subjected to immunoprecipitation and SDS-PAGE (12% acrylamide) under nonreducing conditions. In the case of EGF, Vero cells were used instead of Vero-H cells. The pattern for the whole cell fraction is also shown in lane 4. B, relative radioactivity of fragment A or EGF recovered in the cytosol fraction. The radioactivity was determined by densitometry using a BAS 2000 imaging analyzer. Data are expressed as percentages of the corresponding proteins in nonpermeabilized cells (whole cell fraction).

The cytosol fraction from cells that were allowed to internalize 125I-labeled DT and were then treated with theta -toxin shows a strong band in the position of fragment A, and little or no label in other bands (Fig. 2, lane 2). However, under the same conditions, a small but significant amount of EGF was released into the cytosol fraction, indicating some damage to the endosomes. This could be due to the effects of theta -toxin that entered the cells following disruption of the plasma membrane. To reduce this damage we took advantage of the fact that membrane-bound theta -toxin is relatively resistant to neutralization by exogenous cholesterol. Pronase-treated cells were incubated with theta -toxin at 4 °C for 10 min to bind theta -toxin to the plasma membrane. Unbound theta -toxin was neutralized by addition of serum as a source of cholesterol, and then the cells were incubated at 37 °C for 5 min. Under these conditions about 50% of the fragment A was still recovered in the cytosolic fraction, whereas EGF was not detected (Fig. 2, lane 3). This indicates that fragment A can be recovered from the cytosol under conditions that leave the endosomal membranes intact. Unless indicated otherwise, neutralization of unbound theta -toxin by serum was used in the studies describe below.

Further evidence that the treatment with theta -toxin does not damage endosomal membranes was obtained from experiments in which cells were treated with Pronase a second time after permeabilization. When samples were analyzed by SDS-PAGE, the fragment A band was greatly reduced, but the bands corresponding to full-size DT and the 28-kDa fragment were not affected (Fig. 3). Recovery of EGF was not affected by Pronase treatment of permeabilized cells. When un-permeabilized cells were treated with Pronase, neither fragment A nor other fragments were affected (data not shown). When Triton X-100 was added with Pronase, all protein bands were digested. These results indicate that most of the fragment A molecules are in the cytosol, but full-size DT, the 28-kDa molecules, and EGF are in compartments that are not accessible to Pronase in permeabilized cells. We concluded that the plasma membrane was permeabilized but endosomal membranes were not under the conditions used.


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Fig. 3.   Pronase digestion of cytoplasm in permeabilized cells. Vero-H cells with receptor-bound 125I-labeled intact DT were incubated at 37 °C for 1 h. After treatment with Pronase to remove 125I-DT remaining on the cell surface, cells were permeabilized with C. perfringens theta -toxin. The permeabilized cells were again incubated with or without 10 mg/ml Pronase for 10 min at 37 °C. Pronase treatment was also performed in the presence of 1% Triton X-100. Cell samples were then subjected to immunoprecipitation and SDS-PAGE (12% acrylamide) under nonreducing conditions. Similar experiments were performed with 125I-EGF using Vero cells, except SDS-PAGE was performed in 19% acrylamide gels.

Requirement of Vesicle Acidification for Translocation-- DT requires the acidification of intracellular vesicles to exert its toxicity, and thus various agents that raise vesicle pH inhibit toxicity (25-28, 48). Bafilomycin A1, a specific inhibitor of vacuolar-type H+-ATPase, completely inhibits the toxicity of DT (29). To examine whether the translocation assay described here demonstrates the requirement for vesicle acidification, we tested the effect of bafilomycin A1. Fragment A was not observed in the cytosol fraction when cells were treated with 500 nM bafilomycin A1 (Fig. 4, lane 5), whereas fragment A was translocated to the cytosol in a concomitant control experiment without bafilomycin A1 (Fig. 4, lane 1). These results strengthen the earlier observations that acidification of intracellular vesicles is required for translocation of DT.


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Fig. 4.   Effect of bafilomycin A1 on DT translocation. Vero-H cells were preincubated with or without 500 nM bafilomycin A1 at 37 °C for 30 min. Then, the translocation assay was performed by incubating cells with receptor-bound 125I-labeled intact DT at 37 °C for 1 h before permeabilization. Bafilomycin A1 was present throughout the experiment in the treated cell samples. The cytosol fraction (C) and the whole cell fraction (W) were subjected to immunoprecipitation and SDS-PAGE (12% acrylamide) with (+) or without (-) dithiothreitol.

We used intact DT in this experiment, but most of the DT recovered in the whole cell fraction had been cleaved between fragments A and B, which was demonstrated by SDS-PAGE in the presence of reducing agent (Fig. 4, lane 4). A similar extent of cleavage was observed in the presence of bafilomycin A1 (Fig. 4, lane 8), indicating that acidification is not required for cleavage, consistent with an earlier report (32). In the presence of bafilomycin A1, a significant amount of full-size DT was observed in the cytosol fraction (Fig. 4, lane 5). This full-size DT was also observed in a control sample not treated with theta -toxin (data not shown), indicating that this was not due to the breakage of intracellular vesicles by theta -toxin. A trace amount of DT is recycled from intracellular vesicles to the cell surface, and this recycling of DT seems to be increased in the presence of bafilomycin A1.2 The full-size DT may be from increased recycling of internalized DT in the bafilomycin A1-treated cells during the incubation with theta -toxin.

Time Course of DT Translocation and Effect of Nicking on Translocation Efficiency-- Vero-H cells with receptor-bound nicked or intact 125I-DT were incubated at 37 °C for the times indicated, followed by Pronase treatment and permeabilization. In the case of nicked DT, fragment A was not detected in the cytosol fraction after the 5 min incubation, but a faint band of fragment A was observed at 10 min (Fig. 5). The amount of fragment A in the cytosol increased with longer incubation periods. It should be noted that the permeabilization procedure used includes additional incubations at 37 °C for 5 min for Pronase treatment and 5 min for theta -toxin treatment, thus the total incubation time is 10 min longer than the indicated incubation. It appears that translocation of fragment A into the cytosol takes at least 20 min from the onset of internalization from the plasma membrane.


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Fig. 5.   Time course of DT translocation. Vero-H cells with receptor-bound 125I-DT, in either intact or nicked form, were incubated at 37 °C for the time indicated. The cell membrane was permeabilized, and the cytosol fraction was subjected to immunoprecipitation and nonreducing SDS-PAGE (12% acrylamide). The graph shows the amount of fragment A recovered in the cytosol fraction determined from the intensities of the electrophoresis bands.

Time-dependent translocation of fragment A to the cytosol was also observed with intact DT, but the amount of fragment A translocated was much less than in the case of nicked DT (Fig. 5). After 60 min incubation the amount of fragment A in the cytosol fraction was about 3 times higher for nicked DT than for intact DT. No difference was observed in the rate of internalization of nicked and intact DT from the cell surface (data not shown). Thus, these results indicate that nicked DT is more efficiently translocated than intact DT, at least in Vero-H cells, which is consistent with the toxicities of the two forms for these cells (see Fig. 7).

From the radioactivity of 125I-DT and the intensity of the electrophoresis bands, we estimated the number of fragment A molecules translocated to the cytosol. In the case of nicked DT, about 1.4 × 104 molecules of fragment A were delivered to the cytosol per cell for 1 h incubation. This value corresponds about 5% of the amount of 125I-DT bound to the cell.

Translocation of Fragment A Occurs at an Early Step of Endocytosis-- As shown by time course experiments (Fig. 5), it takes about 20 min from the onset of internalization of receptor-bound DT to the first detection of fragment A in the cytosol. Fig. 5 also shows that fragment A accumulates in the cytosol with increasing incubation time for over 90 min. At least two steps are involved in this accumulation as follows: 1) the internalization step, in which receptor-bound DT is internalized from cell surface into endocytic vesicles, and 2) the translocation step, in which fragment A is translocated from the endocytic vesicle to the cytosol through the vesicle membrane. To separate the translocation step from the internalization step, we carried out a pulse-chase experiment in which translocation of fragment A from the endosomes to the cytosol was observed under conditions in which no additional DT was being internalized.

Cells with receptor-bound DT were incubated at 37 °C for 10 min to allow endocytosis, then the cells were treated with Pronase at 37 °C for 5 min to remove DT remaining on the cell surface and prevent further internalization. Cells were then incubated at 37 °C for the indicated times to allow the translocation of fragment A to the cytosol. Finally, cells were permeabilized, and the amount of fragment A that was translocated to cytosol was determined. As shown in Fig. 6, the amount of fragment A in the cytosol fraction did not increase with time, but was almost constant, and appears to start to decline by 6 min. Since this experiment includes the 10-min endocytosis incubation, a 5-min incubation with Pronase, and 5 min for permeabilization (see time schedule shown in Fig. 6), these results indicate that translocation of fragment A to the cytosol is essentially completed about 20 min after the onset of internalization from the cell surface. In other words, translocation from the endocytic vesicles to the cytosol does not continue very long if newly internalized DT is not supplied to the endosomes.


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Fig. 6.   Pulse-chase study of DT translocation. Vero-H cells with receptor-bound 125I-labeled intact DT were incubated at 37 °C for 10 min. 125I-DT remaining on the cell surface was removed by Pronase treatment, and the cells were further incubated for the time indicated. Then cells were permeabilized, and the cytosol fractions were subjected to immunoprecipitation and SDS-PAGE (12% acrylamide) under nonreducing conditions followed by autoradiography. The intensity of each band was determined using a BAS 2000 imaging analyzer.

In contrast to the translocation step, the internalization step requires more time. In the case of Vero cells, the time required for internalization of half of receptor-bound DT (t1/2) is about 40 min (49),2 while in the case of Vero-H cells t1/2 is about 90 min (data not shown). The prolonged accumulation of fragment A in the cytosol shown in Fig. 6 appears therefore to be due to the prolonged internalization, and translocation of fragment A from the endocytic vesicle to the cytosol apparently takes place in an early stage of endocytosis.

Translocation of Mutant DTs-- To address whether ADP-ribosyltransferase activity is involved in the translocation of DT, mutant proteins with decreased ADP-ribosylation activity were tested. Substitution of serine for Glu-148 within fragment A (E148S) diminishes ADP-ribosyltransferase activity by several hundred-fold (50). E148S and wild-type nicked DT were labeled with 125I, and the translocation activity of each protein was determined. Because the translocation efficiency of DT varies with the degree of nicking, nicked wild-type DT and E148S were used in this study. As shown in Fig. 7A, E148S and wild-type DT had similar translocation efficiencies, indicating that ADP-ribosyltransferase activity is not involved in the translocation of fragment A. 


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Fig. 7.   Translocation and cytotoxicity of mutant DTs. A, translocation assays with nicked wild-type DT and E148S mutant protein. Cells were allowed to internalize receptor-bound 125I-labeled DT or E148S for 1 h at 37 °C before permeabilization. The amounts of fragment A recovered in the cytosol fractions were determined. The rate of translocation of fragment A by E148S was expressed as percentage of the value obtained with nicked wild-type DT. The value represents the average of two independent translocation assays, and bars indicate means ± S.E. B, comparison of translocation activities among DT mutants. Translocation activities of E148S, E349K, D352N, and D352K were determined as in A except that the extent of nicking in E148S was about 45% and in E349K, D352K, and D352K about 60%. The rates of translocation of fragment A by mutant DTs are expressed as a percentage of the value obtained with E148S. The value represents the average of three independent translocation assays, and bars indicate means ± S.D. C, cytotoxicity of mutant DTs. Vero-H cells were incubated with various concentrations of DT at 37 °C for 4 h, followed by incubation with [3H]leucine for 1 h. The radioactivity incorporated into protein was measured. Data are expressed as percentages of protein synthesis by control without DT. The values represent the average of two independent experiments, and bars indicate variations from the means: intact DT (bullet ), nicked DT (open circle ), E148S (×), E349K (black-square), D352N (square ), and D352K (black-triangle).

A number of studies suggest that the T domain within fragment B is important for DT translocation (17, 38, 51). The substitution of lysine for Glu-349 or Asp-352 in the TH8/9 hairpin greatly diminishes both cytotoxicity and low pH-induced pore formation activity in artificial bilayers or intact cell plasma membrane (38). DT mutants E349K, D352N, and D352K were tested in the fragment A translocation assay. Because the mutant proteins used also share the substitution of serine for Glu-148, E148S was used as the control. The translocation efficiencies of E349K and D352K were less than 5% that of E148S, whereas D352N was about 30% of E148S (Fig. 7B). The extent of nicking in the E148S used for this study was about 45%, whereas for E349K, D352K, and D352K it was about 60%. The nicked form of wild-type DT is about 2-3 times more efficient in translocation of fragment A than the intact form, as shown above. Therefore, the relative translocation competencies for E349K, D352N, and D352K are even lower than the values measured here.

To compare the translocation activities of these mutants with their cytotoxicities in the same cell line, inhibition of protein synthesis in Vero-H cells was measured (Fig. 7C). E148S showed a toxicity about a hundred times less than wild-type DT, as previously reported (50). The mutants that share the substitution of Glu-148 to Ser proteins were compared with E148S. Neither E349K nor D352K showed significant toxicity, whereas D352N was about 10 times less effective than E148S. Substitution of Glu-349 or Asp-352 by lysine greatly diminishes both the translocation of fragment A from endocytic vesicles to the cytosol and the toxicity.

Appearance of Novel Fragments of DT in Endocytic Vesicles-- The remnant fraction of permeabilized cells contains DT and DT-derived fragments trapped in endocytic vesicles and other vesicular compartments. Some fragment A that was translocated to the cytosol but is still within the cell pellet would also be contained in this fraction. As mentioned before, a new fragment of DT with an apparent mass of 28 kDa and additional faint fragments of 18 and 35 kDa were observed in addition to full-size DT and fragment A under nonreducing conditions (Fig. 8, lane 1). Since similar amounts of the 28-kDa fragment and the 35-kDa fragment were observed without Pronase treatment (data not shown), these fragments appear to be produced during endocytosis of DT and not as a result of the Pronase treatment. The 28-kDa fragment and the 35-kDa fragment were also observed when Vero cells rather than Vero-H cells were used (data not shown), indicating that the appearance of these fragments is not due to overexpression of DT receptor.


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Fig. 8.   Translocation assay using a reconstituted DT comprising 125I-labeled fragment A and unlabeled fragment B. DT was reconstituted from 125I-labeled fragment A and nonlabeled fragment B (125I-FA-ss-FB) and used in translocation assays. The whole cell fraction was obtained by incubation of Vero-H cells with receptor-bound 125I-labeled nicked DT or 125I-FA-ss-FB at 37 °C for 1 h and subjected to immunoprecipitation and SDS-PAGE.

Under reducing conditions, the 28-kDa band disappeared and the band corresponding to fragment A became more intense (Fig. 8, lane 2), whereas the 35-kDa band was not changed. These results suggest that the 28-kDa fragment includes fragment A, but the 35-kDa fragments does not. To confirm this, DT molecules were reconstituted from 125I-labeled fragment A and unlabeled fragment B (125I-FA-ss-FB). When 125I-FA-ss-FB was used, a labeled 28-kDa band was observed, but the 18-kDa and 35-kDa bands were not observed under nonreducing conditions (Fig. 8, lane 3). The 28-kDa band disappeared, and the fragment A band became more intense after addition of reducing agent (Fig. 8, lane 4). Thus, we conclude that the 28-kDa fragment consists of fragment A and an N-terminal piece of the fragment B, whereas the 35-kDa fragment is derived from fragment B. The size of the 35-kDa fragment suggests that this fragment is the counterpart of the 28-kDa fragment.

As was shown in Fig. 3, the 28-kDa fragment is not affected by treatment of permeabilized cells with Pronase, suggesting that this fragment is inaccessible because it is inside vesicles. Most of the fragment A was digested by this treatment, indicating the fragment A is mainly in the cytosol. To define further the intracellular location of the 28-kDa fragment, subcellular fractionation was carried out in a Percoll gradient. 125I-Labeled transferrin was used as a marker for the endosome fraction. As shown in Fig. 9, the 28-kDa fragment and full-size DT were recovered in the endosome fraction, and fragment A was recovered in top fractions comprising cytosol. The 28-kDa fragment was not recovered in lysosome fractions (determined by activity of lysosomal enzyme beta -hexosaminidase). We conclude that the 28-kDa fragment exists mainly in endosomes.


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Fig. 9.   Subcellular distribution of fragments derived from DT. Vero-H cells with receptor-bound 125I-labeled nicked DT were incubated at 37 °C for 1 h and then incubated for 5 min in 5 mg/ml Pronase solution. The cells were homogenized and subjected to fractionation in a Percoll gradient. Fractions were collected from the top to the bottom of the gradient and analyzed by immunoprecipitation, SDS-PAGE, and autoradiography. The intensities of bands corresponding to full-size DT, the 28-kDa and fragment A were determined. En. and Ly. indicate endosome fractions and lysosome fractions, respectively: full-size DT (square ), 28-kDa (bullet ), and fragment A (triangle ).

The appearance of the 28-kDa fragment was monitored in time course experiments. Vero-H cells with receptor-bound 125I-DT were incubated at 37 °C for the time indicated, and the whole cell fraction was analyzed by SDS-PAGE. As shown in Fig. 10, the 28-kDa fragment was observed even at 5 min after onset of the incubation, and the intensity of the 28-kDa band remained about the same or decreased slightly with prolonged incubation. In contrast, fragment A was scarcely detectable at 5 min and increased with the time of incubation, consistent with the data shown in Fig. 5. Thus, it is clear that the appearance of the 28-kDa fragment precedes that of fragment A. Taken together with the fact that the 28-kDa fragment contains fragment A and exists in the endocytic vesicles, this suggests that the 28-kDa fragment is a translocation intermediate for fragment A of DT.


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Fig. 10.   Appearance of the 28-kDa fragment prior to the appearance of fragment A. Vero-H cells with receptor-bound 125I-labeled nicked DT were incubated at 37 °C for the time indicated. The 125I-DT that remained at the cell surface was removed by Pronase, and then the cells were lysed and the lysate was subjected to immunoprecipitation and SDS-PAGE (12% acrylamide) under nonreducing conditions.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Advantage of the C. perfringens theta -Toxin Permeabilization Method-- Translocation of DT across membranes has been studied using a model membrane (35, 37, 52), an isolated endosome fraction (53), and by low pH-induced direct penetration of the plasma membrane (48, 54). However, little has been reported about the natural translocation process of DT in intact cells, in which fragment A is translocated from the inside of an acidic endosome to the cytosol across a vesicle membrane (55), due to the absence of an appropriate method to observe this process. We have endeavored here to establish a method by which fragment A of DT translocated to the cytosol can be recovered while other fragments remain inside the endocytic vesicles. The most critical step in this method is the selective permeabilization of the plasma membrane without damage to the endocytic vesicle membrane. We conclude that the endocytic vesicle membrane remains intact under the present permeabilization conditions from the following evidence: 1) fragment A was selectively recovered in the cytosol fraction, but full-size DT and other DT-derived fragments were not; 2) EGF was not recovered in the cytosol fraction under the same permeabilization conditions; 3) Pronase treatment after permeabilization of plasma membrane greatly diminished recovery of fragment A, but not other DT-derived fragments and EGF; and 4) the appearance of fragment A in the cytosol fraction was reduced by an inhibitor of vesicular acidification. Thus, the method described here is useful for studying the translocation process of native DT and mutant DTs in intact cells under various cellular conditions.

An advantage of the present method, as compared with previously reported systems, is that the translocation of fragment A proceeds in intact cells, allowing the native process for DT intoxication to be observed. Another advantage is that the translocation assay described here includes all events occurring during DT entry. The entry process of DT includes binding of DT to its receptor, proteolytic cleavage by furin or a furin-like enzyme, internalization of DT from the cell surface to intracellular vesicles, a conformational change of the DT molecule in the acidic vesicle, translocation of fragment A across vesicle membrane, and reduction of inter-fragment disulfide bonds either in the cytosol or in the vesicles. The system described here, which includes all these events, would be useful not only in studying the translocation step but also the internalization and reduction steps.

The method described here may be useful for studies of entry of other toxins, and also for studies of the translocation mechanisms for natural or artificial molecules such as nuclear-locating hormones, growth factors, and immunotoxins. Molecules that penetrate or are translocated across vesicle membranes can be digested by protease or modified by an appropriate reagent from the cytoplasmic face through the permeabilized plasma membrane pores, identifying the molecule or region translocated to the cytosol.

Requirements for and Rates of DT Translocation-- Although DT is cleaved by trypsin or other trypsin-like proteases in vitro (18), experiments using LoVo cells (a cell line deficient in membrane-associated protein furin) showed that furin or furin-like enzymes are responsible for this cleavage in intact cells (32). Nicking of DT with these cellular proteases proceeds partly at the cell surface and partly during endocytosis. In the case of LoVo/Fur1 cells (stable transfectants of furin-deficient LoVo cells ectopically expressing furin), intact DT and nicked DT show similar toxicities. However, in the translocation experiments reported here, nicked DT is about 3 times more efficient than intact DT in releasing fragment A to the cytosol in Vero-H cells, and the cytotoxicity of nicked DT in Vero-H cells is higher than that of intact DT. Higher toxicity of nicked DT has been reported by others (31). This difference may reflect the relative amounts of furin and DTR/pro-HB-EGF expressed on the surface of these cell lines. In the case of LoVo/Fur1 cells, the expression of DTR/pro-HB-EGF is low and a relatively large amount of furin is expressed, and thus DT would be cleaved rapidly, before or after internalization. Because Vero-H cells express a large amount of DTR/pro-HB-EGF molecules on the cell surface and a relatively large amount of DT is internalized into vesicles, the proteolytic cleavage by furin or furin-like enzymes may take longer or be less complete.

Is the nicking process a prerequisite for penetration of fragment A through endocytic vesicle membrane? The fact that the nicked form translocates its fragment A to the cytosol about 3 times more efficiently than intact DT suggests this may be the case. Some reports also suggested the initial cleavage between fragment A and fragment B is followed by carboxypeptidase trimming (56, 57). Further studies using the present permeabilization method would help to clarify these points.

The present system confirmed several observations on the translocation of DT which had not been directly demonstrated for endosomal membranes. The requirement of low pH during the translocation of fragment A to the cytosol has been shown by a number of studies (25, 26, 29, 35, 37, 52, 54, 58). The results presented here confirm that translocation of fragment A from endocytic vesicles to cytosol does not occur when the acidification of vesicles is inhibited by bafilomycin A1.

The cytotoxicities and pH-dependent channel forming activities of E349K and D352K, with mutations in a helical hairpin region of the T domain, were measured previously (38, 40), but translocation from the endocytic vesicles to the cytosol had not been studied. The data presented here clearly show that these mutants cannot translocate fragment A to the cytosol in a natural endocytic process.

Time course experiments showed that fragment A appears in the cytosol fraction within 20 min after the onset of internalization of DT. These results are consistent with the earlier observations from subcellular fractionation studies (55). Although the amount of fragment A in the cytosol of Vero-H cells increased with incubation time for over 90 min if the internalization of DT from cell surface was allowed to continue, such accumulation was not observed if DT bound to the cell surface was removed with protease after a brief period of internalization. These results indicate that translocation from endocytic vesicles to the cytosol takes place soon after endocytosis.

Because previous assays for translocation of fragment A depended on its toxic effect or enzymatic activity for detection, it was not possible to determine whether ADP-ribosylation activity was required. The present study clearly shows it is not required for the translocation of fragment A.

Translocation Intermediates and a Model for DT Translocation-- The T domain is formed of nine alpha -helices (TH1-TH9), arranged in three layers (12, 59). TH8, TH9, and the connecting loop (TL5) form the innermost layer. Two acidic residues, Glu-349 and Asp-352, located on the tip of TL5 have a critical role in the low pH-mediated insertion of the T domain into membranes. Thus, the substitution of Lys for either Glu-349 or Asp-352 reduced translocation through the plasma membrane and the endosome membrane, resulting in reduced toxicity. The second layer consists of three hydrophobic helices, TH5, TH6, and TH7. Although the role of these helices for translocation of fragment A has been less completely characterized, it is likely that they form a channel together with TH8/9 helices through which the fragment A molecule passes when these helices are inserted into the membrane (59).

The third layer consists of three helices, TH1, TH2 and TH3, in the outermost positions. These helices contain a number of charged residues and probably cover the hydrophobic helices, shielding them from the aqueous environment to keep DT water-soluble. Because of the hydrophilic nature of these helices, this domain is not likely to be inserted into a lipid bilayer of the membrane by direct lipid-protein interaction. Several lines of evidence, however, indicate the importance of these helices for translocation of fragment A into the cytosol. Mutation studies of TH1 indicate that the amphipathic structure of this helix, rather than a specific amino acid sequence, is important for determining the cytotoxic activity of DT (60, 61). The mutation of charged amino acid residues to opposite-charged residues or to uncharged residues in TH1 also suggested that TH1 does not interact with the hydrophobic part of the membrane phospholipid (62). These results imply that although TH1 may not take part in the formation of the translocation channel, it has another important role in the translocation process (61).

In this study we found that the 28- and 35-kDa fragments of DT appear in endocytic vesicles. The 28-kDa fragment apparently consists of fragment A and an N-terminal piece of fragment B, whereas the 35-kDa fragment is a part of fragment B and may be the counterpart of the 28-kDa fragment. Judging from the mobility of the 28-kDa fragment in SDS gels, the length of the N-terminal piece of the fragment B in the 28-kDa fragment is about 3 kDa, estimated at about 30 amino acids, which would include the TH1 region of DT. Interestingly, a fragment similar to the 28-kDa fragment was reported to have been produced from receptor-bound DT when cells treated with N-ethylmaleimide, to inhibit reduction of the disulfide bond and release of fragment A from fragment B, were exposed to low pH followed by treatment with Pronase (63). The protease-protected fragment consisted of fragment A and an N-terminal 3-kDa part of fragment B, indicating that TH1 is translocated with fragment A or inserted in the lipid membrane, where it is protected by protease digestion.

Are the 28-kDa fragment and/or the 35-kDa fragment involved in DT translocation? About 30-50% of DT molecules internalized in endocytic vesicles proceed to the translocation of fragment A, but the remaining molecules of DT internalized are degraded in the cells. Although the possibility that the 28- and the 35-kDa fragments are products in the degradation of DT molecules that failed to translocate fragment A to the cytoplasm has not been ruled out, several facts suggest that the 28-kDa fragment is an intermediate in translocation: 1) the 28-kDa fragment is localized in the endosome fraction; 2) the 28-kDa fragment appears before fragment A is detected in the cytosol; and 3) in the time course experiment, the amount of the 28-kDa fragment is constant while the intensity of the fragment A band increases with time.

The T domain of DT appears to consist of at least two functionally separable subdomains. TH8/9 and probably TH5/6/7 form an acid-induced channel in the endocytic membrane which serves as a translocation machinery for fragment A, with or without other membrane proteins, for instance DRAP27/CD9, a diphtheria toxin receptor-associated protein (15, 64). TH1, with or without TH2/3, may serve as a signal or recognition sequence for interaction of fragment A with the transmembrane translocation machinery including TH8/9 and TH5/6/7 at the initial stage of translocation. Proteolytic cleavage to form the 28-kDa fragment may facilitate interactions between these two subdomains. Further studies, for example of mutants that do not generate the 28-kDa fragment, would help clarify whether cleavage to form the 28-kDa fragment is required for translocation.

The translocation assay described here will be useful in further investigation of the roles of the novel fragments and for furthering the understanding of the translocation mechanism of DT and other toxic proteins.

    ACKNOWLEDGEMENT

We thank Dr. Michael R. Moynihan for critical reading of the manuscript.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Tel.: 81-942-376317; Fax: 81-942-313320.

1 The abbreviations used are: DT, diphtheria toxin; EGF, epidermal growth factor; pro-HB-EGF, membrane-anchored form of heparin-binding EGF-like growth factor; DTR, diphtheria toxin receptor; PAGE, polyacrylamide gel electrophoresis.

2 T. Umata and E. Mekada, unpublished observations.

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Abstract
Introduction
Procedures
Results
Discussion
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