From the 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 -helices, whereas the R domain is a
-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 -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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 -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 -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
-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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Translocation Assay Based on Selective Permeabilization of the
Plasma Membrane--
Selective permeabilization of the plasma membrane
using C. perfringens -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
-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.
|
|
|
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.
|
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 -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.
|
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.
|
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.
|
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.
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Advantage of the C. perfringens -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.
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 -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).
![]() |
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|