(Received for publication, April 11, 1995; and in revised form, June 5, 1995)
From the
Active diphtheria toxin consists of two fragments, A and B, joined by a disulfide bond. The B fragment binds to cell surface receptors and aids in the translocation of the enzymatically active A fragment to the cytosol. Normally, the toxin A fragment enters the cytosol from acidic endosomes, but translocation can also be induced at the level of the plasma membrane by exposing cells with surface-bound toxin to low pH. Recently, we showed that disulfide bonds introduced into the A fragment by mutation are inhibitory for translocation. In the present work, we found that although the complete translocation of the A fragment is blocked, three mutant toxins underwent reduction of the interfragment disulfide bond upon low pH exposure, whereas the internal disulfide in the A fragment remained intact. In the case of two of these mutants, the A fragment was released into the extracellular medium upon exposure of cell-bound toxin to low pH. The pH profile for the release of the mutant A fragments was the same as for translocation of wild-type A fragment to the cytosol, and the release was inhibited by conditions that interfere with A fragment translocation. In the case of the third mutant, which remained cell-associated upon reduction of the interfragment disulfide bond, a translocation intermediate was detected. The results show that the reduction of the interfragment disulfide bond can occur in the absence of complete translocation of the A fragment to the cytosol, and they indicate that the reduction takes place at an early stage in the translocation process. Our findings suggest that the translocation of the A fragment across the membrane is initiated at the C terminus.
A number of plant and bacterial protein toxins are able to translocate an enzymatically active moiety to the cytosol of mammalian cells. Once in the cytosol, the enzyme catalyzes the modification of a specific intracellular target, leading to cell death. Most information about the molecular mechanism involved when a toxin crosses the plasma membrane has been obtained in the case of diphtheria toxin. The toxin is secreted as one polypeptide of 58 kDa from Corynebacterium diphtheriae(1) and can be cleaved by low concentrations of trypsin-like proteases into two fragments, A (21 kDa) and B (37 kDa) (2) , held together by a disulfide bond(3) . The B fragment is responsible for binding of the toxin to the specific cell-surface receptor(4) , which is the precursor of heparin-binding epidermal growth factor-like growth factor(5) , and the B fragment also plays an important role in the translocation of the A fragment to the cytosol. Upon receptor binding, the toxin is endocytosed through clathrin-mediated endocytosis(6) , and the acidic environment in the endosomes induces unfolding of the toxin molecule(7, 8) , triggering translocation of the A fragment across the endosomal membrane and into the cytosol(9) . The A fragment catalyzes ADP-ribosylation of elongation factor 2, leading to inhibition of protein synthesis (10) and cell death(11) . If cells with surface-bound diphtheria toxin are exposed to medium of low pH, thereby mimicking the conditions in the endosome, a direct translocation of the A fragment across the plasma membrane is induced(12, 13) , whereas the B fragment inserts into the plasma membrane(14) , forming cation-selective channels(15, 16) . Upon exposure of surface-bound toxin to low pH, two fragments become protected against externally added protease, the entire A fragment, which has reached the cytosol, and a membrane-inserted peptide of 25 kDa, representing the C-terminal part of the B fragment(17) . This assay has been very useful for studying the low pH-induced, direct translocation of the A fragment across the plasma membrane.
Both when the toxin is endocytosed (18) and when translocation is induced by exposure of surface-bound toxin to low pH(19) , the translocation of the A fragment to the cytosol can be observed as a reduction of the disulfide bond linking the A and B fragments together. Although reduction of the interfragment disulfide is a prerequisite for liberation of free A fragment into the cytosol, a reduction of this disulfide bridge does not necessarily implicate that the A fragment has been completely translocated to the cytosol. We reasoned that if the reduction of the interfragment disulfide bond occurs at an early stage in the translocation process, a mutant that cannot be completely translocated may still have its interfragment disulfide bond reduced upon exposure to low pH. In a previous work(20) , we demonstrated that translocation to the cytosol of several mutant A fragments containing an internal disulfide bond was strongly reduced, both when the toxin was endocytosed and when translocation was induced by low pH at the level of the plasma membrane. These data indicate that a substantial degree of unfolding of the A fragment is necessary for translocation to occur. In the present work, we have studied to what extent three of these mutants are translocated partially across the membrane by monitoring cell-mediated disulfide reduction and association of the A fragments with the cells.
Figure 1: Description of mutant A fragments and illustration of the effect of reduction on their migration rates in SDS-PAGE. The positions of the internal disulfide bonds in the mutants CC1 (K24C/D68C), CC2 (N58C/S146C), and CC5 (G119C/N152C) are indicated on a linear representation of the A fragment (A). The relative migration rates in SDS-PAGE of the mutant A fragments in the absence and presence of 20 mM DTT are shown in B. The arrow indicates the position were wild-type A fragment would have migrated. The data in panel B have been published earlier (20) and are here included for the purpose of illustration.
Radiolabeled mutant A fragments were
obtained by in vitro transcription from a T3 promoter,
followed by translation of the mRNA in a reticulocyte lysate in the
presence of [S]methionine. Formation of an
intramolecular disulfide bridge generally increases the mobility of a
protein in SDS-PAGE, and a comparison of the migration rates of the
three mutant A fragments in the presence and absence of reducing agent
is shown in Fig. 1B. All three mutants displayed a
higher mobility in SDS-PAGE in the oxidized than in the reduced state.
Complete toxin was obtained by dialyzing A fragment together with
unlabeled, in vitro made B fragment, allowing the formation of
a disulfide bond between Cys-186 in the A fragment and Cys-201 in the B
fragment(22) . We have previously found that none of the new
cysteines in the mutant A fragments are capable of forming a disulfide
with the B fragment(20) .
When the cells were exposed to buffer of pH 7.0, only
trace amounts of free A fragment were obtained (Fig. 2A). The reconstituted toxin that we used
contained some free A fragment not associated with the B fragment, and
previous work ()indicates that small amounts of free A
fragment bind unspecifically to the plastic of the cell culture plates
at 4 °C. Therefore, we believe that the weak A fragment bands
observed in the case of the pH 7.0 treated cells represent
plastic-bound A fragment being released into the lysis buffer. The A
fragments containing an internal disulfide bridge all migrate faster in
SDS-PAGE in the oxidized state than in the reduced state (Fig. 1B), and in the case of these mutants the small
amount of A fragment associated with the pH 7.0 treated cells was
exclusively in the oxidized state, as judged by their migration rates.
Figure 2:
Low pH-induced cell-mediated reduction of
mutant A fragments. Vero cells were incubated for 1 h at 4 °C with
mutant or wild-type (WT) diphtheria toxin (1 nM),
where only the A fragment was labeled with
[S]methionine. The cells were washed three times
with PBS to remove unbound toxin and subsequently incubated for 3 min
at 37 °C with MES-gluconate buffer of pH 4.8 or 7.0. The
MES-gluconate buffer was recovered, and total protein was precipitated
with trichloroacetic acid and analyzed by SDS-PAGE and fluorography (panels D and E). In some cases (panels A and B) the cells were lysed directly after the incubation
with MES-gluconate buffer, whereas in others (panel C), they
were incubated for 5 min at 37 °C with HEPES medium containing 5
mg/ml Pronase E and 10 µM monensin. The cells were
detached from the plastic by the treatment and were transferred to an
Eppendorf tube and washed in HEPES medium containing 1 mM phenylmethylsulfonyl fluoride and 1 mM NEM. The cells
were lysed in lysis buffer for 10 min at 0 °C, and proteins in the
post-nuclear supernatant were precipitated with trichloroacetic acid
and analyzed by non-reducing SDS-PAGE and fluorography. Arrows indicate migration rates of wild-type diphtheria toxin A fragment (A) and of the complete toxin (DT).
When the cells were exposed to pH 4.8, the A fragment bands were substantially stronger than at pH 7 (Fig. 2B). Also, in case of all three mutants (CC1, CC2 and CC5), the A fragment was primarily in the oxidized state, and the A fragment bands were generally weaker than for wild-type toxin.
To distinguish between reduction of the interfragment disulfide bond and complete translocation of the A fragment, a parallel experiment was carried out where the cells were treated with Pronase after the low pH treatment to remove non-translocated material. In this case, the oxidized forms of the mutants CC1, CC2, and CC5 were not visible on the gel, whereas the wild-type A fragment was not digested by the Pronase (Fig. 2C).
Although the oxidized form of all the mutant A fragments was liberated from the B fragment upon low pH treatment, it was not protected against externally added Pronase. This indicates that although an internal disulfide in the A fragment does not prevent low pH-induced cell-mediated reduction of the interfragment disulfide bond, it prevents complete translocation of the A fragment to the cytosol, in agreement with our previous findings(20) .
Since only the B fragment is responsible for binding of the toxin to the cells(4) , it is conceivable that reduction of the interfragment bond of surface-bound toxin containing a non-translocatable A fragment may lead to release of A fragment into the extracellular solution. Therefore, we also subjected the trichloroacetic acid-precipitable material from the pH 7.0 and 4.8 buffers to SDS-PAGE and fluorography. The results showed that substantial amounts of the mutants CC2 and CC5 were present in the pH 4.8 buffer (Fig. 2E) but not in the buffer of pH 7.0 (Fig. 2D). The material in the medium migrated as expected for mutant A fragments with intact intrafragment disulfide bonds. The observation that the mutants CC2 and CC5 were released into the medium upon reduction of the interfragment disulfide bond indicates that the cell-mediated reduction of the A fragment occurs at an early point in the translocation process, when the A fragment is not yet tightly associated with the cells by virtue of being at an advanced stage in the process of translocation.
Figure 3: Pronase-protected translocation intermediate of CC1. The experiment was similar to that in Fig. 2C, but the gel was exposed longer. Only the low molecular weight range is shown.
Figure 4:
Comparison of pH profiles for release of
mutant A fragments into the buffer and translocation of wild-type A
fragment to the cytosol. Mutant or wild-type (WT) diphtheria
toxin, labeled with [S]methionine only in the A
fragment, was bound to Vero cells by incubation for 1 h at 4 °C.
Unbound toxin was washed away, and the cells were exposed to
MES-gluconate buffer of various pH values. Protein in the MES-gluconate
buffer was precipitated with trichloroacetic acid, and the cells were
treated with Pronase, washed, and lysed in lysis buffer. Protein in the
post-nuclear supernatant was precipitated with trichloroacetic acid.
Finally, the precipitated material was analyzed by SDS-PAGE and
fluorography.
Figure 5:
Inhibition of release of mutant A
fragments and translocation of wild-type A fragment. Diphtheria toxin,
labeled with [S]methionine only in the A
fragment, was bound to Vero cells, unbound toxin was washed away, and
the cells were incubated for 15 min at 37 °C in HEPES medium
containing 10 µM monensin and either 100 µM NEM, 20 mM sodium acetate, pH 5.5, 100 µM Cibacron Blue, 100 µM DIDS or no addition. Then, the
cells were incubated for 3 min at 37 °C in MES-gluconate buffer, pH
4.8, containing the same additions (20 mM sodium acetate at pH
4.8, in the case of the acetate treatment). In the case of the mutant A
fragments (CC2 and CC5), total protein from the MES-gluconate buffer
was precipitated with trichloroacetic acid and analyzed by SDS-PAGE and
fluorography (two upper panels). In the case of wild-type
toxin, the treatment with MES-gluconate buffer was followed by Pronase
treatment to remove non-translocated material. Subsequently, the cells
were washed and lysed, and proteins in the post-nuclear supernatant
were precipitated with trichloroacetic acid and analyzed by SDS-PAGE
and fluorography (lower panel).
Figure 6:
In vitro reduction of inter- and
intrafragment disulfide bonds with DTT. In vitro translated,
[S]methionine-labeled mutant A fragments (CC1,
CC2, CC5) were dialyzed overnight against dialysis buffer to allow
formation of the intrachain disulfide bond. Wild-type,
[
S]methionine labeled A fragment was dialyzed
together with unlabeled B fragment to form full-length toxin
(A-S-S-B). The proteins were incubated for 15 min at
25 °C in HEPES medium containing the indicated concentrations of
DTT. NEM at a final concentration of 100 mM was added to
quench excess DTT, and after 2 min, SDS-PAGE sample buffer was added,
and the samples were subjected to non-reducing SDS-PAGE and
fluorography (A). The arrows indicate migration rates
of full-length diphtheria toxin (DT), wild-type A fragment (A), and the oxidized (A
) and
reduced (A
) forms of the mutant A
fragments. B, the bands representing the A fragments in their
reduced states on the gels in A were quantified using a
scanning densitometer, and the percentage of A fragment in the reduced
state is expressed, assuming the highest value obtained for each
protein to correspond to 100%.
, CC1;
, CC2;
, CC5;
+, A-S-S-B.
The experiment in Fig. 6was carried out at neutral pH, and since the cell-mediated reduction of diphtheria toxin is induced at low pH, it would be more appropriate to do the experiment at low pH. However, we found that the reduction with DTT was very inefficient at pH 4.8, and the four disulfides in question were reduced only to a very low extent even at the highest concentrations of DTT tested (data not shown).
Figure 7:
Cell-mediated reduction of endocytosed
mutant toxin. Reconstituted mutant or wild-type diphtheria toxin,
labeled with [S]methionine only in the A
fragment, was bound to Vero cells for 1 h at 4 °C. The cells were
washed four times in PBS and were subsequently incubated for increasing
time periods at 37 °C in HEPES medium. The cells were subsequently
lysed, and protein from the post-nuclear supernatant was precipitated
with trichloroacetic acid and analyzed by non-reducing SDS-PAGE and
fluorography. In the case of the mutants CC1, CC2, and CC5, the arrow (A) indicates the migration rate of the
oxidized form of the A fragment. WT, wild type; DT,
diptheria toxin.
When the endocytosis experiment in Fig. 7was carried out in the presence of a mixture of three inhibitors of lysosomal degradation (pepstatin, leupeptin, and antipain), the oxidized forms of the mutants CC1, CC2, and CC5 disappeared less rapidly (data not shown), indicating that the liberated, non-translocated A fragments are broken down in lysosomes.
The most important observation in this study is that diphtheria toxin mutants containing non-translocatable A fragments may undergo cell-mediated reduction of the interfragment disulfide bond upon exposure to low pH. Two of the mutant A fragments were released into the low pH medium, a release which was inhibited by several compounds known to inhibit diphtheria toxin translocation. These data show that the reduction of the interfragment disulfide bond can occur in the absence of complete translocation of the A fragment to the cytosol, and they indicate that the reduction occurs at an early stage in the translocation process.
We favor a model (Fig. 8) for the low pH-induced translocation of the A fragment to the cytosol where membrane translocation is initiated by the insertion of the transmembrane domain of the B fragment, pulling into the cytosol the N-terminal part of the B fragment and the C-terminal part of the A fragment. The interfragment disulfide bridge is then reduced upon exposure to the reducing conditions in the cytosol. This is supported by other studies suggesting that the C-terminal part of the A fragment (29) and the N-terminal part of the B fragment (30, 31) are important for A fragment translocation. Although the introduced disulfides in the A fragment are capable of efficiently blocking the complete translocation of the A fragment to the cytosol, they do evidently allow the A fragment to unfold sufficiently to let the interfragment disulfide bridge reach the cytosol. Interestingly, CC5, which is almost completely released into the medium, has its internal disulfide between positions 119 and 152 and therefore more close to the interfragment disulfide (position 186-201) than that in CC1 (between positions 24 and 68), which becomes stuck in the membrane, possibly because a large part of the A fragment is translocated before the process is stopped by the intrafragment disulfide (Fig. 8). CC2 (disulfides between positions 58 and 146) exhibits an intermediate situation. Consequently, the presented data suggest that the translocation of the A fragment starts at the C terminus.
Figure 8: Hypothetical model for membrane translocation of diphtheria toxin A fragment. The toxin binds to the cell-surface receptor by its B fragment. Exposure to low pH leads to the insertion of the B fragment into the membrane and partial translocation of the A fragment across the membrane, leading to reduction of the interfragment disulfide bond through exposure to the reducing environment of the cytosol. This step can be inhibited by the indicated compounds. The complete translocation of the A fragment can be blocked by internal disulfide bonds, leading either to release of A fragment into the medium or to non-translocated A fragment stuck in the membrane.
If CC1 is translocated from the C terminus
until the most C-terminal cysteine (Cys-68) of the intrafragment
disulfide bond is encountered, a translocation intermediate of 13 kDa
should be obtained after Pronase treatment to remove external material.
Instead, a translocation intermediate of apparent molecular mass of
11.5 kDa was observed, indicating that translocation may have been
arrested approximately 15 residues C-terminal to Cys-68. Since the
amount of translocation intermediate observed in the case of CC1 was
smaller than the amount of CC1 generated upon cell-mediated reduction
of the interfragment disulfide bond, it may be that translocation is
halted at several different stages and that the observed 11.5-kDa
intermediate represents the most frequent point of arrest.
A number of compounds reported to interfere with the translocation of the wild-type A fragment to the cytosol also blocked the low pH-induced release of the mutants CC2 and CC5 into the buffer. Such treatments have been shown to affect toxin translocation in different ways. In the presence of NEM, partial translocation of the A fragment occurs, leading to the formation of a translocation intermediate(26) , whereas insertion of the B fragment into the plasma membrane and formation of cation channels are only moderately inhibited(17, 26) . DIDS inhibits both translocation of the A fragment (32) and insertion of the B fragment into the plasma membrane(17) , while acidification of the cytosol inhibits A fragment translocation (17) and formation of cation channels (16) but has no apparent effect on the insertion of B fragment (17) . Treatment of the cells with NEM is likely to quench cellular reducing agents, and it is therefore not surprising that NEM inhibited the release of CC2 and CC5.
Incubation of the cells with sodium acetate, pH 5.5, leads to acidification of the cytosol through diffusion of membrane-permeant acetic acid molecules through the plasma membrane(33) . We consider it less likely that the observed inhibition of the release of CC2 and CC5 by sodium acetate, pH 5.5, is due to effects other than acidification of the cytosol, since treatment with pH 5.5 in the absence of sodium acetate had no inhibitory effect. There are two possible ways by which acidification of the cytosol may inhibit the cell-mediated reduction of the interfragment disulfide bond. One possibility is that the reducing ability of the cytosol may be abrogated by lowering the pH, similarly to what we observed in the case of in vitro reduction with DTT. Alternatively, dissipation of the transmembrane proton gradient may remove the driving force necessary for translocation. In the case of the complete translocation of wild-type A fragment, acidification of the cytosol is likely to exert its inhibitory effect through removing a driving force, because if its action were only on the level of interfering with the reduction of the interfragment disulfide bond, one would expect to observe a translocation intermediate similar to that seen in the NEM-treated cells. However, in the case of the release of the mutants CC2 and CC5, the inhibition by acidification of the cytosol may well be on the level of inhibiting cytosolic reduction of the interfragment disulfide, since this release may require translocation of only a short segment of the A fragment, and this partial translocation may not have the same requirement for a proton gradient as the complete translocation of the A fragment to the cytosol.
At
neutral pH, the intrafragment disulfide bonds in the mutant A fragments
were in general much more easily reduced by DTT than the interfragment
disulfide bridge. At pH 4.8, the pH value used to induce translocation,
DTT was inefficient in reducing both kinds of disulfide bonds, and we
were therefore unable to address the relative reducibility of the
disulfides at low pH. In the case of reducing thiols such as DTT and
glutathione, it is the thiolate anion that is the active form, and the
amount of thiolate relative to thiol at a certain pH value is
determined by the pK value, which is in
the range of 9-10 for DTT and glutathione(34) . Thus, the
concentration of thiolate is very low at low pH values, and this may
kinetically limit reduction reactions that are thermodynamically
favorable.
It has been suggested that the cell-mediated reduction of the interfragment disulfide bond of diphtheria toxin is mediated by a general reductive function at the plasma membrane(27) , possibly by a cell-surface protein disulfide isomerase(28, 35) . Our data argue against this since we observed extensive cell-mediated, low pH-induced reduction of the interfragment bond, which was quite resistant toward reduction with DTT, whereas the intrafragment disulfide bond, which is in general more sensitive toward reduction with DTT, was not reduced.
The observed release of the mutant A fragments CC2 and CC5 into the low pH medium suggests that these A fragments can undergo partial translocation, sufficient to reduce the interfragment disulfide bond, followed by a reversal of the translocation, leaving the A fragment in the extracellular medium. Such reversal of translocation processes has been observed in several other translocation systems. A truncated form of bactericidal/permeability-increasing protein, generated in a cell-free system from a transcript lacking a stop codon, can be translocated into microsomes, but it is unable to be retained, leading to retrograde movement of the protein out of the microsomes(36) . The translocation of the precursor protein proOmpA into inner membrane vesicles from E. coli can be reversed when factors necessary for complete translocation are removed(37) . Similarly, the complete translocation into the mitochondrial matrix of a fusion protein consisting of a presequence for import into mitochondria fused to dihydrofolate reductase could be inhibited by stabilizing the structure of dihydrofolate reductase through methotrexate binding, and although the presequence could be cleaved by a matrix peptidase, the processed protein was recovered in the supernatant, indicating reversal of translocation(38) .
Studies with model membranes have shown that both the A and B fragments of diphtheria toxin can insert into lipid bilayers at low pH(25, 39) , and this insertion in the absence of a receptor has been suggested to reflect the mechanism of A fragment translocation in the living cell. The results from the present study suggest that the A fragment is quite loosely associated with the membrane during the initial phase of translocation, since the non-translocatable A fragments CC2 and CC5 can be released from the cells after reduction of the interfragment disulfide bond.