Ion channel and membrane translocation of diphtheria toxin

FEMS Microbiology Immunology 105 (1992) 101-112 © 1992 Federation of European Microbiological Societies 0920-8534/92/$05.00 Published by Elsevier

101

FEMSIM 00236

Ion channel and membrane translocation of diphtheria toxin

Cesare Montecucco, Emanue le Papini, Giampietro Schiavo, Elisabetta Padovan and Ornella Rosset to

Centro CNR Biomembrane and Istituto di Patologia Generale, Universitd di Padova, Padova, Italy

Key words: Diphtheria toxin; Membrane translocation; Conformational change; Ion channel

1. SUMMARY

Diphtheria toxin is the best studied member of a family of bacterial protein toxins which act inside cells. To reach their cytoplasmic targets, these toxins, which include tetanus and botulinum neurotoxins and anthrax toxin, have to cross the hydrophobic membrane barrier. All of them have been shown to form ion channels across planar lipid bilayer and, in the case of diphtheria toxin, also in the plasma membrane of cells. A relation between the ion channel and the process of membrane translocation has been suggested and two different models have been put forward to account for these phenomena. The two models are discussed on the basis of the available experimental evidence and in terms of the focal points of difference, amenable to further experimental investigations.

2. INTRODUCTION

Diphtheria toxin (DT) is secreted by toxigenic strains of Corynebacterium diphtheriae and it is

Correspondence to: C. Montecucco, Istituto di Patologia Gen- erale, Via Trieste, 75, 35121 Padova, Italy.

responsible for most clinical manifestations of diphtheria [1,2]. DT is the most studied member of a large family of bacterial protein toxins that act inside cells on cytoplasmic targets [3]. They share a similar mechanism of cell intoxication that can be conveniently divided into three steps: a) binding; b) membrane translocation; and c) target modification. This functional similarity is reflected in an overall similar structural organiza- tion. In fact these toxins are composed of two protomers generally held together by a single disulfide bond: protomer A has a catalytic activity while protomer B is responsible for cell binding and translocation into the cell [3]. The reduced toxin or the isolated protomers are non-toxic and toxicity can be recovered by reforming the disulfide link. Most of these toxins display an ADP- ribosyltransferase activity, i.e. they transfer ADP-ribose from cytoplasmic NAD to a more or less specific cytoplasmic target [3,4]. This is the case of DT, exotoxin A from Pseudomonas aeruginosa, cholera toxin, pertussis toxin and the C2 and C3 clostridial toxins. Shighella shigae and Shiga-like toxins are rRNA-adenine glycohydro- lases [5]. Tetanus and botulinum neurotoxins are zinc endopeptidases [6,7].

DT is secreted as an inactive single polypeptide chain which is later activated by protease cleavage at an exposed loop [2], as schematized in

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Fig. 1. The A chain (21164 Da) ADP-ribosylates specifically elongation factor 2 at a single residue, a modified histidine, termed diphtamide. This results in the blockage of protein synthesis and cell death by apoptosis [2,4]. The B chain (37194 Da) binds to a protein receptor via its 6-kDa carboxy-terminal part [8,9]. Cell sensitivity to DT is correlated to the amount of its receptor: the very sensitive Vero cell line displays tens of thou- sand receptors per cell [10]. The DT-receptor complex is collected into coated pits and endocy- tosed. Drugs which interfere with the acidification of the endosomal lumen by the ATPase proton pump also reduce or prevent cell intoxication [11-14]. Moreover DT can be induced to enter into cells from the plasma membrane by a short incubation in an acid medium, thus by-pass- ing endocytosis [15-17]. These experiments have indicated that acidification is a prerequisite for the entry of DT into ceils. Moreover, the endo- some by-pass procedure is very important because it allows one to test in vivo several parame- ters that may influence the phenomenon [18].

The discovery that a simple condition such as acid pH is sufficient to cause a rather remarkable phenomenon such as the translocation of a

~c**.

PROTEASE$

( 21 kDa )'NH2 A s1 ,s

( 37 kDa ) - C ~ H S

Fig. 1. Schematic structure of diphtheria toxin. The toxin is secreted as a single inactive polypeptide chain. Several proteases can cleave the chain at an exposed loop thus generating an active toxin composed of two subunits held together by a single interchain disulfide bond. Protomer B is responsible for cell binding and membrane translocation while protomer A is an ADP-ribosyltransferase specific for elongation factor two

of eukaryotes and arehaebacteria.

water-soluble protein across the hydrophobic membrane barrier has triggered a wealth of studies aimed at a molecular understanding of such a process. Most of them were performed on model membrane systems employing a variety Of biophysical and biochemical techniques. Attention was also paid to compare model and cellular experimental systems [19].

3. THE ION CHANNEL OF DIPHTHERIA TOXIN IN PLANAR LIPID BILAYERS

By applying DT to planar lipid bilayers at different pH values it was found that at low pH DT and the B fragment of the truncated mutant crm 45 (lacking the 16.5-kDa carboxy-terminus) form voltage-dependent ion channels [20-25] with the following main properties: 1) the DT channel has a low conductance, few picoSiemens (pS) at physiological ionic strength; 2) the DT-induced increase in conductance of the membrane occurs only at low pH and does not require a transmembrane pH gradient, though the rate of increase of conductance is higher with a pH gradient. With no pH gradient across the planar lipid bilayer a DT-induced current is seen only in the presence of an applied voltage; voltage is not needed when a pH gradient is present; 3) the channel is cation selective at pH 5.5; 4) more than one toxin molecule, possibly two, participate in the formation of a channel; 5) the conductance of planar lipid bilayers containing acidic phospholipids in the presence of DT at low pH is higher than those made only of zwitterionic lipids; and 6) the rate of conductance increase is strongly dependent on the existence of a positive potential (posi~ tive on the c/s side where DT is present).

Analysis of the change of permeability induced by DT on potassium-loaded liposomes led to es- sentially the same conclusions [26,27]. These studies have been very important because, apart from introducing the use of model membrane systems, they provided a clear indication that DT undergoes a pH-driven structural change and that, as a consequence, at low pH it interacts deeply with the lipid bilayer.

4. T H E T U N N E L M O D E L FOR T H E MEM- BRANE TRANSLOCATION OF DIPHTHE- RIA TOXIN

Another important aspect of these biophysical studies was that they provided a first experimental support for the tunnel mechanism of DT translocation across membranes proposed by Bo- quet et al. [28] on the basis of detergent binding experiments. Moreover, this model identified protons as a single agent capable of inducing the membrane interaction of DT. The main features of the tunnel model are schematically summarized in Fig. 2. Fragment B was suggested to undergo a conformational change from a neutral form to an acidic form, characterized by the presence of hydrophobic surfaces which mediate detergent and lipid binding. The acid form of fragment B is thus able to insert into lipid bilayers forming a transmembrane hydrophilic pore large enough to allow the passage of the unfolded A chain. The protein pore, probably made of two B polypeptide chains, protects subunit A from the interaction with lipids. In this model it is assumed that ions flow through the same channel used by the unfolded A chain to transverse the membrane and the formation of the channel is a prerequisite for A translocation to occur. On the basis of this model one may expect that a single

(9 ®

~ acid ic pH ,ons

Fig. 2. The tunnel model for the membrane translocation of diphtheria toxin. At acid pH fragment B becomes hydrophobic and inserts in the lipid bilayer in an oligomeric form with a hydrophilic pore. Fragment A unfolds and crosses the membrane inside the tunnel protected from the contact with lipids. When free of the A chain, the protomer B tunnel is an ion

channel.

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pore incorporated in a planar lipid bilayer is able to mediate the passage of more than one A chain. In the tunnel model the A and B subunits of DT are seen as two independent domains with respect to the conformational change triggered by pH: protomer B is suggested to become hydrophobic and to insert into the lipid bilayer while protomer A is suggested to unfold before inserting into the channel.

5. T H E ION CH A N N EL OF D I P H T H E R I A TOXIN IN CELLS

Some aspects of the ion channel made by DT on planar lipid bilayers left the possibility open that the ion channel could be a peculiarity of this model system and, even if not so, that the channel could be non-relevant to the translocation of D T across membranes. For example, as appears from Fig. 2, its pH dependence is shifted at least one pH unit toward lower pH values with respect to that of cell intoxication [18,29]. Another con- troversial aspect concerns the size of the channel. Though there is no direct relation between the size of a channel and its conductance, values of the order of few picoSiemens do not fit with the dimensions expected for a protein channel that has to accommodate a polypeptide chain with its lateral chains of different volume, charge and hydrophilicity. This consideration is the more valid in the light of the properties of the first protein-conducting channel characterized in planar lipid bilayers [30]. Simon and Blobel [30] have shown that this channel of the rough endoplasmic reticulum is closed when plugged by the nascent polypeptide chain and is opened by puromycin, which induces the release of the peptidyl- puromycin complex. The channel shows a large conductance (220 picoSiemens) with no ion selectivity. The change in size or polarity of the applied voltage does not influence either the conductance or the gating of this channel. These properties are those expected for a pore that has to accommodate amino acids with lateral chain differing in size, charge and polarity.

To obtain evidence for the formation of a DT channel on living cells, Vero cells in culture were

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exposed to DT in an acid medium for various time periods. Sandvig and Olsnes measured the cellular uptake of various radioactive ions [31], Papini et al. measured the K ÷ and Na ÷ cellular content [32] and Alder et al. measured leakage of radioactive metabolites [33]. The following conclusions were reached: a) at low pH DT forms an ion channel on the plasma membrane; b) its pH dependence is almost superimposable to that of cellular intoxication; c) the channel is specific for monovalent cations, does not leak out amino acids or phosphorylated metabolites and does not take up glucosamine; d) the membrane remains per- meable to monovalent cations as long as a transmembrane pH gradient is maintained; on return- ing the extracellular medium to neutrality, the cell quickly recovers its normal K ÷ and Na ÷ content; and e) the channel itself does not con- tribute significantly to the death of the cell because a DT mutant lacking ADP-ribosylating activity forms the channel but does not kill the cells.

Taken together, these studies on the permeability induced by DT on cells have also provided strong evidence for the formation of a DT ion channel on ceils. However, some differences among channels formed on planar membranes and on cell membranes are evident, as summarized in Table 1. In particular there is a different pH dependence and an apparent different size. These differences can be ascribed to the presence in the cellular system, but not in the model systems, of the toxin receptor. Unfortunately no

single channel patch clamp studies of DT on cells have been reported so far and any further comparison is not possible.

6. THE LOW pH-INDUCED INSERTION OF DIPHTHERIA TOXIN INTO THE LIPID BILAYER

The structural changes of DT-induced by pH in relation to model membranes have been studied with a variety of techniques including liposome binding [34], membrane photolabelling with photoreactive lipids [35-37] and phospholipids [38,39], circular dichroism [36,40], tryptophan flu- orescence [41,42], liposome fusion and leakage [42-46], scanning calorimetry [47], polarized infrared spectroscopy [48] and lipid monolayers [49]. Notwithstanding the differences among the experimental approaches, these studies show a sub- stantial agreement on the main findings with a good correlation with results obtained with cellular systems. The more relevant conclusions, de- rived from the above-quoted studies, can be summarized as follows:

(a) At neutral pH, DT interacts with the surface of negatively charged membranes. The possible relevance of this membrane surface interaction of DT to cell surface binding has been discussed elsewhere [39].

(b) A downshift of pH induces a true structural change in DT from a neutral conformation

Table 1

Comparison of the properties of the channels made by diphtheria toxin planar lipid bilayers and in Vero cells with those of a protein-conducting channel of the endoplasmic reticulum

Property DT in planar DT in Vero cells Protein- lipid bilayers conducting channel

of ER

Conductance Few ps n.d. 220 pS Estimated few pS

Ion selectivity X + X + Non-selective Size of permeant ion > Glucosamine < Glucosamine > Gluconate pH Dependence < 5.0 5-6 n.d. Voltage gating + n.d. - Voltage dependence of conductance + n.d. -

n.d. = not determined.

to an acidic conformation. The two forms differ in their secondary structures and exposure of tryptophan residues. While the neutral form is water-soluble, the acidic form aggregates as a result of the exposure of previously hidden hydrophobic regions. The hydrophobicity of acid DT makes it able to penetrate into the hydrophobic core of lipid micelles or bilayers with a strong preference for negatively charged lipids.

(c) Both the A and B fragments of DT are involved in the low pH-induced interaction of the toxin with the fatty acid portion of phospholipids: such interaction extends all along the hydrocarbon chains. In this process, a-helices of the inner part of B chain orientate parallel to the lipid acyl chains, while B-sheets of the carboxy terminal part of the B chain orient parallel to the membrane surface. While the low pH-induced lipid interaction was predicted by the tunnel model, the finding that A also interacts with the hydrophobic lipid core of the membrane at low pH was unexpected.

(d) The low pH-induced lipid interaction of chain A is fully reversible, i.e. chain A appears to insert in the lipid bilayer at low pH and to re- escape when pH is brought back to neutrality.

(e) The neutral and acid structures of DT have different shapes with different apparent molecular areas; 2950 A 2 at pH 7.4 and 3450 A 2 at pH 5.0. The lipid-embedded part of DT at pH 5.0 has an apparent molecular area of 2100 A 2.

(f) The decrease of pH brings about the exposure of the interchain disulfide bond, which becomes accessible to reduction by thioreductase [50].

(g) The transition between the neutral and acid forms of DT in the presence of negatively charged liposomes occurs between pH 5 and 6 in close agreement with the pH dependence of DT cell intoxication and ion channel formation, shown in Fig. 2, and at variance from that obtained in planar lipid membranes.

(h) Chain B is the first part of DT that inserts into the membrane and this B insertion is not the rate-limiting step of DT membrane translocation.

(i) The membrane translocation competent form of DT is likely to be a multimer-aggregated toxin.

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7. A CLEFT MODEL FOR THE MEMBRANE TRANSLOCATION OF DIPHTHERIA TOXIN

Some well-documented experimental results such as the low pH lipid interaction of fragment A and the reduced size of the channel formed by DT in living cells, cannot be accommodated in the tunnel model of DT membrane translocation. Moreover, as discussed above and summarized in Table 1, the properties of the recently characterized protein-conducting channel of the endoplasmic reticulum are very different from those of DT [30].

At this stage one is left with two possibilities: (1) the ion channel formed by DT at acid pH is not directly involved in toxin membrane translocation. Such an epiphenomenon would easily explain the above-mentioned experimental results; and (2) the DT ion channel is involved in the membrane translocation step, but there are differences among the fragment A-conducting and the ion-conducting forms of DT.

As the large majority of researchers in this field, we have adopted as working hypothesis the latter one. However, to account for all the experimental findings and theoretical considerations summarized above, we have put forward a 'cleft'

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-.: -i o _ " ~ -

~ 8 '~

° -

• •

O 4 5

pH

Fig. 3. pH Dependence of diphtheria toxin block of Vero cells protein synthesis ( • ), ion channel formation in Vero cells (~)

and in planar lipid bilayers (e).

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model for the m e m b r a n e t ranslocat ion of D T [19,51], as schemat ized in Fig. 3. We propose that toxin cell binding is the result o f the interactions of D T with bo th a prote in receptor and the headgroups of negatively charged phospholipids [39]. Experimental evidence for such a double interaction and its biological implications are discussed elsewhere [39]. The D T protein receptor is a 14-20-kDa glycoprotein which binds D T via its polypept ide chain [7-9]. A n addit ional prote in may be involved in toxin binding [52]. The 6-kDa carboxy-terminal region of p ro tomer B is mainly responsible for the interact ion with the prote in receptor [9].

Recep to r -bound D T is internalized via coated pits and it is collected into endosomes. As a result of the acidification o f the endosomal lumen, D T adopts an acidic structure, characterized by the exposure of hydrophobic regions, including the interchain disulfide bond, previously

h idden in the prote in interior. Such hydrophobic surfaces can be shielded f rom the contact with water ei ther by involvement in p ro t e in -p ro t e in contacts or by insertion into the hydrophobic core of the lipid bilayer. Even though there are no data on the stoichiometry of the membrane- in- serting form of p ro tomer B, it is likely to be a dimer or a multimer, whose format ion is driven by hydrophobic segments exposed at acidic pH. A multimeric organizat ion of the D T channel is also suggested by the generally oligomeric structure of the known ion channels and by considerat ions on the number of f ragment B segments candidate for the insertion in the lipid bilayer.

The apparen t p K a of m e m b r a n e insertion of p ro tomer B is about 6. On this basis the protona- tion of histidine residues (15 out of the 16 such residues present in D T are clustered in chain B) is expected to play a major role in its conformational transit ion together with prolines, which

(9 ® @

low ~

neutral neutral ~ pH pH )

tow

C Y T O P L A S M

Fig. 4. A cleft model for the membrane translocation of diphtheria toxin. I. DT binds to the cell surface of DT:sensitive cells via a protein-protein interaction with its glycoprotein receptor (R) and via electrostatic interactions with negatively charged lipids. The COOH-terminal region of chain B is responsible for binding R, while its amino-terminal part is kept near the carboxy-terminus of protomer A by the interchain disulfide bond, hidden in the hydrophobic protein interior. II. At low pH DT changes conformation. In a monomeric or multimeric form, alone or together with R, chain B forms a hydrophilic cleft that wraps the hydrophilic surfaces of protomer A during its membrane penetration while the hydrophobic surfaces of chain A are exposed to the lipids. Some toxin charges are neutralized during membrane translocation by counterions of phospholipid head groups or small anions. A matching of hydrophilic (protein-protein and protein-water) and hydrophobic (protein-protein and protein-lipid) interactions is required to render the process energetically feasible. The first part of chain A that crosses the membrane is its carboxy-terminus linked to the amino-terminus of chain B with the exposure of the interchain disulfide to the cytosol. The neutral cytosolic pH and/or the transmembrane potential induce the refolding of protomer A to its neutral and catalytically active form. III. While the now water-soluble chain A leaves the hydrophilic wrapping cleft, the margins of the cleft come together in order to minimize the energetically unfavorable interactions with lipids. However, this leaves a transmembrane alignment of hydrophilic residues that

constitutes a transmembrane ion channel. Due to its reduced size, this channel can accommodate only small ions.

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may undergo cis-trans isomerization as proposed by Brasseur et al. [53].

The central proposition of the cleft model is that, during the membrane penetration of DT, fragment B forms a hydrophilic cleft that drives the insertion of chain A with its hydrophobic segments exposed to lipids and their hydrophilic regions interacting with corresponding regions of the hydrophilic cleft of fragment B. Such a matching of hydrophobic and hydrophilic protein-lipid and protein-protein interactions is of fundamental importance in making the translocation process energetically feasible.

The cleft model can accommodate the possibility that the DT receptor participates in the membrane translocation of protomer A [54] by assum- ing that together with protomer B it forms the hydrophilic cleft that nests chain A during its passage through the membrane; it is also possible that the efficiency of the chain-A-conducting channel made of protomer B plus DT receptor is higher than that formed by fragment B alone in model membranes, but that both structures are able to function as protein channels.

The disulfide-linked C-terminal part of A and N-terminal part of B are suggested to be the first portion of DT that inserts in the membrane to reach the cytoplasm, mainly driven by the hydrophobicity of the disulfide bond. These two terminal segments are expected to play a crucial role in the process and thus not to tolerate insert- ions, deletions or substitutions altering their phys- ico-chemical properties. Indeed, it has recently been shown that DT mutants extended at the carboxy-terminus of chain are less or non-toxic [55].

The A chain crosses the membrane with its hydrophilic residues facing the protein channel and its hydrophobic lateral chains exposed to the hydrocarbon chains of lipids. The open structure of the B cleft can accommodate the different bulkinesses of different portions of the A chain. Moreover, such a transmembrane structure is expected to be able to translocate across the membrane peptide segments added to the N-terminus of the A chain, but not to the C-terminus, as recently found by Stenmark et al. [55]. It should be emphasized that no unfolding of A is required

by the cleft model. Rather it is suggested that A undergoes a conformational change in concert with B. The low pH structure of A is amphipathic with mainly hydrophilic surfaces inside the protomer B cleft and an hydrophobic side interacting with lipids.

The interchain disulfide bond, previously hidden in the hydrophobic protein interior, becomes exposed on the other side of the membrane to the cytoplasm, where it is ready to be cleaved either by chemical or proteic reducing agents of yet unknown localization [50].

It is likely that the membrane penetrating form of DT has a net positive charge and that some Lys, Arg or His residues are not in the position to form salt bridges with corresponding negatively charged Asp and Glu residues. Due to the high energetic cost of driving charged groups across the hydrophobic core of the lipid bilayer (particu- larly the positive charges of lysines and arginines), membrane insertion and translocation of DT would be made less energetically unfavorable if these charges were neutralized and shielded by formation of ionic couples with negatively charged phospholipid head groups or small anions such as chloride, bromide or thiocyanate. This effect would help to explain the finding that anions are involved in the penetration of DT into ceils and that the more hydrophobic membrane permeant anions are the more efficient [56]. It is possible that the membrane-translocating form of DT is actually a transient anion-phospholipid-multimeric toxin complex that assembles on the low pH lumen side of the endosomal membrane and lasts until it reaches the cytoplasmic face of the membrane, where the complex disassembles because its ion couples are released by water solva- tion. Such a proposal emphasizes the role of non-bilayer lipidic configurations [57,58], though direct evidence for such structures in DT membrane translocation is lacking. However, this sug- gestion is in agreement with the well-documented ability of DT to induce fusion of lipid vesicles at low pH that is believed to occur via zones ot DT-induced lipid destabilization [44].

At the present stage it is hard to envisage how the polypeptide chain of protomer B folds across the membrane to form the hydrophilic cleft and

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interacts with lipids at the same time. The only available information is that Lys 299 and Glu 248 and 249 are located on the face opposite to the cytosol [59]. Moreover, the loss of channel activity of DT on replacing I1e-364 with a Lys residue [60] suggests that I1e-364 is essential for the correct assembly of the channel.

The finding that chain A can escape from liposomes once pH is returned to neutrality suggests that, after reduction of the interchain disulfide bond, fragment A can re-acquire its neutral water-soluble form on facing the neutral cytoplasmic pH [38].

A comparison of the number of molecules of DT bound to a cell with the estimated number of A chains in the cytoplasm has indicated that the membrane-translocation step of DT is a ineffi- cient process [50]. However, in experiments aimed at testing the possible role of the endosomal proteases in the cellular intoxication of DT (Papini et al., in preparation), it was found that the translocation and release in the cytosol of the catalytic chain A subunit is a rather efficient process that occurs in competition with a proteolysis of DT stimulated by the acidification of the endosomal compartment. In other words, the A chains in the cytoplasm are those that have es- caped endosomal proteolysis by translocation in the cytosol. Endosomal proteolysis lowers the overall efficiency of intoxication and is responsible for the previous underestimation of the efficiency of membrane translocation that is per se higher than previously thought. At the present stage, the proposal that endosomal proteolysis generates a B45-1ike fragment which is the form of B actually responsible for A translocation is still to be tested [28]. The fact that endosomal proteolysis is avoided in cells intoxicated with DT by exposure to a low pH extracellular medium [17,50] leaves open the possibility that a Bas-like fragment is generated by proteolysis in the endosomal lumen and it mediates a more efficient translocation of A with respect to that occurring at the plasma membrane with the intact B fragment.

The cleft model suggests that, after chain A has reached the cytoplasm, the margins of the hydrophilic cleft embedded in the lipid bilayer,

formed by protomer B (or protomer B plus DT receptor), come together to reduce to a minimum the amount of hydrophilic protein surface exposed to the hydrocarbon chains of lipids. This leaves a transmembrane alignment of hydrophilic residues that constitute a peculiar fiat-shaped ion channel with more or less rigid protein walls and a very flexible lipid side. Such a channel is expected to have a low conductance because of its reduced size and at the same time to be able to accommodate large charged objects provided that they have a hydrophobic fiat portion such as an aromatic ring. Such features would account for the finding that the DT channel on cells has a low permeability to choline, glucosamine, amino acids and phosphorylated intermediates [31-33].

In this simplified model the major driving force for the membrane insertion and translocation of DT is the transmembrane proton gradient because it is the low pH that causes the conformational transition on one side of the membrane and it is the neutral pH that induces the refolding of chain A on the other side of the membrane. However, a contribution of membrane potential is suggested by cellular studies [61,62]. Moreover, the voltage opening of DT channels at low pH with no transmembrane pH gradient [20-25] suggests that voltage is able to unplug the channel by inducing the release of the A chain. Also, the role of disulfide reduction in membrane translocation and in the refolding of protomer A in the cytoplasm remains to be investigated.

The tunnel model and the cleft model differ in several respects. The main differences amenable to experimental testing can be summarized as follows: 1) the low pH-driven conformational change of DT. In the tunnel model the A and B protomers are supposed to change conformation independently: B becomes more hydrophobic and inserts in the lipid bilayer forming a pore, while A is suggested to unfold in order to be able to enter in the pore. In the cleft model the conformational change induced by acid pH is seen as a concerted phenomenon involving B and A at the same time. There is no experimental evidence for an unfolding of A at low pH, rather it appears that the catalytic fragment of DT adopts an acidic conformation that can be crystallized and charac-

ter ized spectroscopically [48,63,64]; 2) lipid interaction. In the tunnel model f ragment A crosses the m e m b r a n e inside an hydrophil ic pore with no contact with lipids. The model does not explain how the 65 hydrophobic residues of the A chain can get th rough an hydrophil ic pore, part icularly the 20 Trp, Tyr and Phe residues. In the cleft model the m e m b r a n e t ranslocat ion of f ragment A occurs at the lipid prote in boundary in a way compat ible with the different proper t ies of the lateral chains of the different amino acids; 3) proper t ies o f the D T ion channel. In the tunnel model the channel that mediates the transloca- t ion of A is also the one that carries ions across the membrane : the prote in conduct ing channel and the ion channel are the same entity with the same dimensions. The format ion of the tunnel is actually a prerequisi te for the m e m b r a n e translo- cat ion of f ragment A. In the cleft model the ion channel is re lated to the s tructure that has mediated the t ranslocat ion of A, but it is not the same entity, not having the same size and propert ies; the ion channel is a consequence of the membrane t ranslocat ion of A. This difference is such that the tunnel model would be dismissed by the finding of a muta t ion or of an agent that allows cell intoxication without channel formation.

8. M E M B R A N E T R A N S L O C A T I O N O F O T H E R B A C T E R I A L P R O T E I N T O X I N S

Much less is known about the mechanism of cell entry of o ther bacterial prote in toxins with cytosolic targets such as the exotoxin A f rom Pseudomonas aeruginosa or the clostridial neurotoxins of te tanus and botulism, Diphther ia toxin has been the toxin pro to type in the study of all aspects o f cell intoxication. It is therefore likely that some aspects of its mechan i sm of act ion are relevant to those of o ther toxins. Several biophysical and biochemical studies indicate that these toxins also undergo an acid-driven structural t ransit ion with exposure o f hydrophobic surfaces and consequent interact ion with the hydrocarbon chains of lipids [65-74].

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A C K N O W L E D G E M E N T S

We thank Drs. R. Bisson, G. Menes t r ina and D. Pie t robon for critical reading of the manuscr ipt and we acknowledge the suppor t of the ' C N R Target Project for Biotechnology and Bioinstru- mentat ion ' .

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Ion channel and membrane translocation of diphtheria toxin

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