Biochirmca et Bioph),sica Acta. I I 13 ( 1992 ) ~ - 5 I © t992 Elsev;er Science Publishers B.V. All rights reserved (}.~4 4157/92/$05.00

B B A R E V 85398

Diphtheria toxin: membrane interaction and membrane translocation Erwin London D e t r i m e n t of Biochemistry" and Cell BitdoRy, State Umtxorur), o[ New Yc~rk at .~ito~l.vBrook, Ston~" Bn)ok. IVY (USA~ (Received .1 September I ( ~ I )

Contents I.

Intr~.,x:luction: bacterial protein toxin,, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2fl

II.

Diphtheria toxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2t~

III.

Struclurc of diphthct ~ toxin anti its fragm::nts in the native ~,t;fl: . . . . . . . . . . . . . . . . . . . . . .

27

IV.

Diphtheria toxin hydrophobicity and membrane in.'~'rtion at low pH . . . . . . . . . . . . . . . . . . . .

28

V.

(.'onformational changes in diphtheria toxin at k~v pH . . . . . . . . . . . . . . . . . . . . .

),4}

VI.

Why Io~, p l l :.Iter~ diphtheria folding and hydrophobicity

34

VII.

Pot,.. form:alton b:, dlphthetia toxin: bilay(r lipid membrane sludie~ . . . . . . . . . . . . . . . . . . .

3~

VIII.

Pore f,.Irmation: vc~,icle studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

37

IX

Pore forma.on in cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.'~

X.

The cffecl ,ff lip,d struglute on inlera~.'lio~ ~,'ith diphtheria toxin and the origin of elcctroslatic effect,, on in~rtion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

38

XI,

Ink'racti,m ot na[i~,e diphlheP.i toxin v, ilh Jipids at neulral pll . . . . . . . . . . . . . . . . . . . . . . . .

39

"ql

Toxin mducLd membrane fusion and membrane aggregat.)n . . . . . . . . . . . . . . . . . . . . . . . .

40

XIII

Toxin ~If-aggn:gation and oligomcr lormatam . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

40

XIV.

(.'l,:a~,age of the link b,:tween lhc A and B domai.~, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

41

XV.

Role of toxin disulfide 1.4*nd~,in cntr~,' into cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

41

XVI,

Membrane interaction and conformation of natural mutants of diphtheria toxin . . . . . . . . . . .

42

...........................

AbhrcviatiGas: ApUp. adcnyly143',5'I-uridinc-Y-pho.~phatc; TINS, 2-p-toluidinylnapthalcnc-b-sulfonatc; BLM. bilaycr lipid membranes; CD, circular dichroism; DPA. dipicolinic acid; DSC, differential .~anning calorimetry; D T r . dithiothreitol; EF-2. elongation factor 2: IHP, inositol hexapht~,phate: IR infrared; L U V , large unilamellar vesicle.,,; M L V , muhilamcllar vesicles; NBD. N47-nitrobcnz.2-oxa-l.3.diazol-4.~l); PC, phosphatidykholinc; PG, phosphatidylglycerol; PIP, phosphatidylinositol; SDS. sodium dodccyl sulfate: SUV. small unilamellar vesicles; TD,

34 trifluoRrmet hyl ). 34 m-iodophenyl )diazirinc. OJrr~'slxmdence: E London, Department of Biochemistry and Cell Biology, State University of New York at Stony Brook. Stony Br~x)k, NY 11794-5215, USA.

26 XVII.

I)iphlhcr:a toxin lr;,;,,,h~:ation acros,, mevJcl and cell membranes

.....................

43

X V I I I . Diphlhcria hlxin receptor: idcnlit~ and ~s~ihlc inllucncc on Ioxln behavior . . . . . . . . . . . . .

46

XIX.

M(vJ¢l for translocalion ol diphtheria I*~xin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4h

XX.

('ompari~m of ,'he behavior ,~! P~'eudomrma~cXOlC.~xinA to Iha! of diphtheria toxin . . . . . . . . .

47

XXI.

Parallels, holy, con Ioxin membrane Iran,~location and Ihc Iranshv.:alion o f ordinaD' cellular proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

48

Rcrnaining queslion,, and fulur¢ rc,,varch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

48

XXII.

Acknov, lcdgcmcn{ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

48

Rclcrcncc,. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

48

I. Introduction: bacterial proteip toxins

Pathogenic bacteria often produce protein toxins that I~S,~ss enzymatic activities. "l'hc.~ toxins have been of interest fiw ovcz one hundred years. They have the ability to disrupt cells by interfering with a single step in cellular metabeli*~m. Often. they inhibit protein synthesis. In other cases they alter signal transduction aertrss membranes. Because of these actions they play an im~)ttant role in bacterial disea.~, and they are often the key virulence factors in bacterial infection. In fact, an intensive effort to combat bacterial diseases by developing more effective anti-toxin vaccines still continues [I]. It should also be noted that thcir potential as agents of biological warfare is a concern in our pre~nt day wolld. Recently. toxins have begun to play a medically u~ful role in the fi~rm of immunotoxms, lmmunotoxins are artificial toxin-antibody hybrids designed to specifically destroy cell types to which their antit~ly moictie,~ bind. If targeted against a particular type of diseased cell thcy can act as highly specific and potent therapeutic agents [2.3]. Presently, immunotoxins are the subject o f intensive research in such areas as cancer, autoimmune disease and AIDS [3.5]. Bacterial protein toxins are also of interest as bit)chemical tools. For example, the ability of some toxins to modify ,.;-proteins has helped dissect the relationship r~tween transmembrane signal transduction and cell function [6]. Toxins are also proving useful for elucidating the mechanism of protein translocation across membranes becau.~ they must cross membranes to gain entry to the cytoplasm. Diphtheria toxin is perhaps the most well understood of the membrane-translocating toxins. The purpose of this review is to summarize what we have learned about how this toxin is able to accomplish translocation, with an emphasis on studies at the biochemical level. Several reviews have covered the enzyrnolug,y -,,id gc,ictic~, of the toxin [7-11]. There have

al,~ becn rcvicws that have considered the cellular t ranslocation process [ 12- Ih]. The interested reader is urged to consuh these for the viewpoints t~f ,,.,~mc of the other investigators in this field. I!. Diphtheria toxin

Diphtheria toxin is produced by Coqwehacterium diphtheriae, the bacterium that c a u l s the d i ~ a ~ diphtheria. It is a medium sized protein (M, 58348) that is secreted as a single polypeptide, but can be divided into two domains, chains or fragments, A (M, 21 167) and B (M, 37 199) [l(I]. Since the A and B fragments appear to be distinct folding domains (see section V) they will bc rc~'erred to as domains when part of whole toxin and fragments when considered in isolated form. However, it should be kept in mind their folding may not be totally independent of each other The toxin enters cells by receptor-mediated endocytosis and then penetrates the membrane of an acidic organeUc, most probably an endt~.,~m=c [17-19]. After membrane penetration st is behevcd that the catalytic A fragment is released into the cytoplasm. Once in the ~topla~m. the A fragment is able to ADP-ritx~sylate protein synthesis elongation factor 2 (EF-2) [7]. In this reaction the ADP-ribose portion of NAD* is transferred to diphthamide, a i'x)sttranslationally mt~ified tits unique to EF-2 [7]. An ()utlinc of the entry process is shown in Fig. I. The ADP-ribosylation of diphthamide inactivates EF-2, thereby inhibiting protein synthesis and causing cell death. It has recently been suggested by one group that the toxin can induce intcrnuclcosomal DNA breakdown, and that it has a nuclcase activity which may bc rcsponsiblc for this proccss [20-23]. However. this proposal is controversial because a contaminating nuelea~ has been identified in ~ m c preparations of the toxin [24,25]. Several groups have determined the amino acid sequence of the toxin through gene [26,28] and amino acid sequencing [29,30]. The protein is synthesized as a

27 ......

.............

\ 1 81ndlng

~¢o*

~.¢ **,-*-,o*

\'

; EF 2

•

:

i

~

~

Endoeytosis

/"

'"

~.-" + ' : ~,

2

A ~ •

~"/

i

s I.~,b,t.o~ o,

":-~'

\ ~ !/

/

3. Aci,* Triggered

Change

.~

':i..

/

/ ' I ~x -

__--

4 Insertmon

(~ ;A S I,i . i .1._ i/ .

~/

Fig. I. ,%hcmatic outline of Ihr: ~.tcp~of toxin entry into ¢c11.,. Reprintedhy pt-rmi.~ionfrom Ref. ~7.

precursor containing a 32 residue N-terminal signal ~quencc. The mature protein contains 535 amino acids, 193 in fragment A, which forms the N-terminal third of the protein, and 342 in fragment B, which forms the C-terminal two thirds. The A and B domains are connected by a proteinase sensitive link. However, proteolytic cleavage between A and B domains ('nicking') is insufficient to allow their dissociation because they are also held together by a disulfide bond between C'ys-186 and 201. There is aim a disulfide bond betwcen C-'ys-461 and 471 in the B domain. The most notable feature of the primary structure of the toxin is a ~ r i e s of relatively hydrophobie regions within the B domain. No such hydrophobie regions have been observed within the A domain. After the initial ~quencing of the CNBr fragments of the B domain it was recognized that the CBI fragment (residues 340-459) centain.~ significantly hydrophobic regions [31]. It was also noted that the N-,.crminal part of tifi,~ sequence could form an :amphipathic helix. Another possible amphipathic helix was p r o p o ~ d in the N-terminus of the B domain (in the CB4 pcptide), but this region seems rather hydrophilic. Aftcr the complete sequencing of the toxin, hydrophobic regions could be specified more complctely. The regions lacking charged residues arc 265-289: 3(10-317: 328-348; 353-371 (which has one charged residue) arid 424-439. These correspond to the most hydrophobic regions on a hydropathy plot [26.32] and may form transmembrant (x-helices, although they contain ,some polar residues. In one prediction .';chcme the region covering residues between about 360-370 fit a surface (amphipathic) helix [32]. This sequence is part of the segment predictcd to be amphipathic in CB]. The sequence of the B domain has lent support to i.he view that it pTays the major role in the membraneinsertion process. However. we shall sec that this conclusion is deceptively simple.

i l l Structure of diphtheria toxin and its fragments in the native state In the native state at pH 7 diphtheria toxin behaves like an ordinary water soluble prot::in. It appears that the hydrophobic stretches within the B domain are buried under these conditions. The secondary s'.ructure of the toxin is not remarkable, as both IR and circular dichroism (CD) indicate the toxin has a mixture of a-helix and ,B-sheet structure [33-35]. The tertiary structure is not known although progregs has been made towards ,solving a crystal structure. However, the homology between the catalytic do;~ahi~ ,,~" Fseudomonas cxotoxin A and diphtheria toxin allows us to extrapolate to ,some degree from the structure of the exotoxin (see below). Additional information about native toxin structure has been derived from its interaction with various ligands, in the native state the whole toxin has the ability to bind N A D + tightly and catalyze its hydrolysis [36]. This suggests that the A domain must b¢ largely folded properly within the whole toxin. However, whole toxin cannot catalyze the ADP-rit~sylation reaction, oo~sibly duc to steric blockage of EF-2 binding by the B domain. An unusual feature of the native toxin is the ability to bind the dinucleotide ApUp extremely tightly [37], and a large fraction of purified toxin molecules usually contain bound ApUp [38,39]. ApUp binds at the N A D * binding site [40]. However, the cationic (.'-terminal portion of the B domain is also involved in tight binding of ApUp and is responsible for the binding of other anionic ligands such as IHP [41,42]. The region on the B domain involved in ligand binding has been termed the P-site. The P-site is believed to be spatially adjacent to the N A D + binding pocket of the A domain [42,43]. Reccptor binding also involves the C-terminal region of the B domain, and is restricted to the most C-terminal 6 kD;, fragment [44,45]. There is evndencc suggesting that ~he re,:eptor and P-sites overlap to some dcgrcc from the inhibition of Ix)th rcccptor binding and toxicity by P-site ligands such as ATP and IHP [41.46]. In contrast, although ApUp can reduce toxicity [47] the toxin can bind to it and the receptor at the same time [48]. One early proposal was that the cadonic receptor site directly interacts with a receptor molecule that is anionic [41,43,49], but this has not been confirmed yet. it should also be noted that there is one report implicating the central portion of the B domain in receptor binding [50]. This conclusion is based upon the indirect evidence of neutralization of toxicity by monoclonal antibodies. At pH 7 the toxin can be found either as a monomer or dimer [51.52]. Dimer formation tends to occur upon freezing in phosphate buffer [53l. The monomer and dimer differ in that the dimer is much le~ toxic to cells, probably because dimerization prevents binding

28 to the toxin receptor [32]. One very curious, fcatt, re of toxin behavior is the extremely low rate of i :tcrconversion between monomer and dimer [52]. To explain this it has been suggested that the dimer might be held together by exposed hydrophobic contacts [52]. ttowever. the spectro~opic and conformational properties of monomer and dimer are very similar, suggesting they have the same overall structure [34,52]. The A fragment of the toxin can be relee,:,ed from whole toxin by proteolysis and reduction of the A-B disulfide bond. In isolated form the A fragment is able to catalyze ADP-ribosylation 154]. At neutral pH it ,'-2h3v~ as a soluble hvdroohilic proh'in I!~ ~hC77Aai unfidding occurs c~x~peratively as expected fl~r a globu lar protein [55.56]. it is extremely resistant to irreversible denaturation [9.56]. CD suggests it is low in a-helix content [33] whercas IR studies indicate that it has mixed a-helix and B-sheet slructurc 1351. 1"he analogy to the homologous catalytic domain of cxotoxin A [57-62] would, based on the cxotoxin crystal structure, ptt:dict a globular structure compo~ed of mixed a-helix and B-sheets with a cleft that accommodates N A D ' [62,63]. The behavior of the imflated B fragment is not well characterized. Most studies have examined B fragment i~lated under denaturing conditions and with its internal disulfide reduced. After removal of denaturant it is somewhat hydrophobic at neutral pH, and it has only been rei'~rted to be soluble in borate buffer [~4.65]. It has not been shown that isolated B fragment prepared in this w~.y takes on a cooperatively foldt:d conlor,na!ion similar to that it has in whole toxin. IR indic,Its that the i~flated B fragment does contain some secondary structure composed of mixed a-helix and /'Jsheet [35]. In addition, i,,~lated fragment B has bccn reported to possess the ability to compete with toxin for binding to cellular receptors [h4,60]. However. it is not clear whether this would require native structure because cvcn a pcptidc fragment of the B domain retains receptor binding [45]. In addition, the isolated B fragment dt~s show increased hydrophobicity at low pH [67]. but this certainly does not pr:wc the native structure is present at neutral pH because cvcn unfolded toxin shows pH-dcpcndent hydrophobicity (see section V). Furthermore. since wc shall see that toxinmembrane interaction involves a denaturation-like process it would not be surprising that partially unfolded fragment B retains appropriate hydrophobic behavior. On the other hand the fact that isolated B fragment can be combined with the A fragment to form native toxin does suggest it can take on the native conformation under some conditions [66.68]. More definitive indications that the isolated B fragment can properly fold comes from tb¢ properties of a deletion mutant (called B36) missing the A domain [691. B36 forms pores with the .same specificity as whole toxin, and

:;haws pH-dependent confi~rmational changes that are similar to those seen with whole toxin (see section V). B36 may fold properly because it is not expand to denaturing conditions during its preparation. It would be interesting to know the status of the internal disulfide in this molecule. I.~ss of the internal B domain disulfide can have severe effects on toxin behavior fsee section XVI).

IV. Diphtheria toxin hydrophobicity and membrane insertion at low pH 3"he binding of Triton X-Ill() to the toxin at neutral pH provided the first hints as to how it might cross a membrane [70]. It was found that denaturation of whole toxin with a low concentration of ~ d i u m d~u.lccyl sulfiitc (SDS) induced the eXlX~.~urcof Triton X-I(l(I binding ,,,ites. However. Triton X-I~MI was not bound by the isolated A fragment under any condition,:. The observation thai the C-terminal truncated CRM 45 mut'mt wa,: able to hind detergent without denaturation suggested a m(uJcl in which there was a proteolytic processing event exit)sing hydrophobic regions. This supported a mt~el in which there were hydrophobic regions on the N-terminal half of the B domain that w e r e normally buried. It was al.~) proposed that the B fragment might fi)rm a pore through which the A fr:,gm,:a; could pass (Fig. 2 (top)). T h e unusual property of the isolated A fragmc,lt to readily rcnature suggested a m~vJel in which the A domain pas~d through a pore m unfifldcd fi)rm and then refi)ldcd in the cytoplasm. The obscrva!;on by two groups that endtv:y'tosis and low pH were critical for the entry of the toxin into the cytoplasm was a major advance [17,18]. (Experimentally, it in the ability to use low pH to trigger membrane insertion where and when desired that has allowed dissection of the translocation process.)"l"hesc investigators were able to demonstrate the role of low pH by showing that agents that raise pH in acidic vacuoles prevented inhibition of protein synthesis and by showing that after bkK'king endt~ytosis, transfer of cells to a low pH medium was able to induce the toxin to cross the plasma membrane and inhibit protein synthesis. They concluded the ent.,'v process it,valved endemytosing of the toxin into the lumen of an acidic vacuole where the low pH triggered translocation. A number of studies have confirmed these reports, including not only the reports of in vitro properties demribed in detail below, but also the observations that cells with a defect in vacuole acidification are resistant to diphtheria toxin [71-74], and that inhibitors of vacuolar H" pumps inhibit toxicity [75]. Other studies have confirmed that the toxin reaches acidic vacuoles via endocytosis [19,76,77]. Given the pH

2~

1976, 81

~98a

1984-5

t987-8

~

b

c

d

:, TA~ ,, ~ ~.A:_~ /

L,,

T>

.

~

R,,

~r'C

-,

T >.~7~C

%

."

8 • N ~HI I , 4~*C,

~'~,! ~]

L'

T
. Unfolding of the toxin and its domains has also hccn studied using differential ,~anning calorimctr~ (DSC) [55,107] At neutral pH it was found that thermal unfolding of toxin monu0ncrs without I'~und ApUp was consistent with two domains irreversit~'~ unfl)lding at 55 and 58°C [55]. From the magnitude ot the cnthalpv changes unfolding of the A domain was assigned to the 55°C transition. A residuz., thermal unfolding event could be detected upon ."eating toxin at low pH. However, the emhalpy change upon unfolding was found to be much reduced, and had the cooperativity characteristic of a single ok)main. The magnitude of the enth'ilpy change suggested that at lower temperature low pH unfolded the B domain and heating at low' pH would unfo'0d the A domain Proteolysis studies have provided additional evidence that the contorm.,tional change at low pH is accompanied by increased exposure of new sites. As

noted ahoy,c, toxin exposed to low pH and then returned to neutral pH showed an increased susceptibility to trypsin digestion [82]. A later study reported that this increased sensitivity t.o trypsin could also be det,'.cted when digestion was performed at low pH [89]. Thc sites of ir.crcascd digestion were located within the N-terminal region of the B domain, implying that this region became exl~scd to solution at low pH. It was also found that the trypsin sensitive site in the A domain (involving cleavage at Lys-39 [60]) was digested less at low pH than at neutral pH. However, this may he a consequence of the marked d e c r c a ~ in the intrinsic activity of trypsin at low pH. Incrca~d digestion of t:)xin at low pH can also be observed usipg chymotrypsin and protcinase K (Calhoun, R. and London. E., unpublished oh.~n'ations). Information about the secondary structure of membrane-inserted toxin at low pH has been obtained by IR spectroscopy using an attenuated total reflection method [35,109]. In whole toxin membrane-insertion at low pH induced a decrease in a-helix and a correSl~mding increase in an anomalous signal assigned to a low frequency/3-sheet relative to the structure at neutral pFI. Because of the lack of information on IR performance for membrane proteins this conclusion nust be considered tentative. In the case of bacteriohodopsin apparent D-sheet. identified by IR was not confirmed by the high rest)lution structure Ill0,111]. On the other hand, an unusual secondary structure could he an important factor in membrane insertion by the toxin. Changes in fragment A ur,:m low pH exposure and membrane insertion were sirfilar to those in whole toxin. The change in fragment A structure was reversed when pH was returned to pH 7. in agreement with the reversibility of other properties [56]. IR detected no change in fragment B folding at low pH. but as noted in section Ill. isolated fragment B may not take on its native state conformation. The CBI region of fragment B showed an increase in (~-helix at low pH. The helices appeared to he oriented parallel to the lipid a(.)'l chains. It was al.~ found that there were changes in a small amount ol .oxin that I~)und to membranes at pH 7. it seemed to have higher helical coatent than in ~ l u t i o n The state of folding in m,.mhranc-inserted toxin was al.~) examined in Ref. 112. After low pH treatment. two conformations were found, one which predominates at lower temperatures and another more unfolded form which predominates at higher temperatures, including at 37°C. Proteol~sis and activity measurements showed that the A domain was unfolded in the higher temperature form. Using the higher temperature form it was found that after pH neutralization and cleavage of the A-B disulfide the refolding and reactivation of the A domain occurred and it was released from the membran,':. This was consistent with

33

the proposal that A domain unfolding and rcfolding play a role in tnmslocation (see section XIX). By comparing the amount of fluorescence quenching of toxin by phospholipids spin-labeled at deep and shallow positions within the membranc [1(14] it was found that the toxin penetrated deeply into the membrane in both low and high temperature form~ [!12]. However. this result and the related crosslinklng results noted earlier [85,86] should be t,,te~preted cautiously as we have no idea to what degree the toxin perturbs the packing of lipids in its local environment. Membrane penetration of low pH treated toxin changed significantly after pH neutralization [I 12]. The membrane-inserted toxin did not return to the native state upon pH neutralization. However, in agreement with photolabeling results [85], les,,,cr fluoresc,,mce quenching suggested that there was a decrease in the degree of insertion in the bilayer, even though the toxin remained membrane bound. This was true for both low and high temperature conformations. One interpretation of the effect of pH neutralizatior is that it causes the toxin to protrude from the membrane. An alternate possibility is that it causes the membrane-inserted toxin to cluster so as to exclude lipids laterally. Although this latter cxplanation cannot be ruled out yet, we ,see no increase in oligomer size upon pH neutralization in preliminary experiments (see section XIII). Fig. 2 (bottom) summarizes the various conformations taken on by mc:nbrane-inserted toxin as defined in Ref. 112. Recently. it has been shown that denaturation of the toxin at high pH induces behavior very similar to that at low oH [!13]. At high pH ( > 10.5) there is an increased exposure of Trp ces~dues to solution, as judged by fluoreseencc, and an increased hydrophobicity, as judged from the ability of the tox;n to bind to detergent micclles and model membrane vesicles. As the temperature is increased at high pH there is a second cooperative unfolding transition. Preliminary studies with isolated A and B fragments suggest that the A domain unfolds in the first high pH denaturation step and the B domain in the second. Howevcr. this conclusion is tentative because of the possible cffccts of interactions between A and B domains and the difficulty of working with the isolated B fragment noted earlier. Based on the pH and temperature dependence of the conformation of the A and B domains, the folding of the toxin under different conditions can be illustrated by the phase diagram in Fig. 3. It is noteworthy that the precise conditions of the experiment can have a significant effect upon the structure of the toxin when close to the conditions likely to occur in the endosome, 37°C and pH 5-5.5. In another recent study topographical information about plasma membrane bound toxin was derived from

T 50; 0 uJ r'r" :::::)

H,,

A d B~

A d Bd

;

A. B.

40

H, i

A, B,

r'r" LL/

~B,

.i

~= 30 I.-.-

2oiI_. . . . . ./,,_t. . . . .B., ...... 5

!_.

.L . . . . L. . . . . .

7

pH

9

~'1

Fig 3. p i t - t h e r m a l phase (or s t a t e ) d i a g r a m for diphth,:ria toxin, l"h,s d i a g r a m identific:,, h)xm c~mfi~rmulion at any p i t / t e m p e r a t u r e combination. N is the nati~,¢ state. I. arc I(~, o i l conformations,. It are high ptl confi~rmations. T is the thermally .Jenaturcd conli~rmation i;I neulral pll. The R conformations formed when toxin in an L state is exposed to neutral p | t arc not shovvn (see Fig. 2). When a domain is folded it is denoted by f . W h e n a d o m a i n b, at ]east partly unfuIdcd it i:, denoted by 'd" ('denatured'). Ek,undarles arc ~,h~wvn between confl)rmations s e p a r a t e d by cts~pcralive folding transitions. T h e exact ~a~sition o f a houndaD' d e p e n d s on incubation time and fi~rm of to~in u,,cd. Notice th~t fi~r unfi)ldcd prolcin, p i t changes only appear h) cau~ mm-ctmpcralivc confi~rmati~mal change,.. Such conformati, mal changes arc expected because of the ioni/=lion of Asp. Glu. and | i t s ='esidues (in the p l ! 4 - 7 ran Re) and o f T y r J n d Lys residues., (in the pit 9 - I I ) range.

proteolysis experiments [ii4]. In these experiments proteolysis sites were Iocaiized from the comparigm of the size of the digcstio, pr,~ucts to fhc size of the subfragmentation products obtained ~ith iodosobcnzoie acid or hydroxylamine. Incorporation of [~sS]Cys in various fragments confirmed the digestion sites. From digestion by externally added proteinase VH, pronase and trypsir sites cxl~)sed to the external aqueous environmc.t wcrc identified to be at or near G!u-240.241 or 24~,249 (usi.g proteinasc vg), Lys-264 and Lys-29v ~usi.g ~.ypsin) and Lys-299 (using pronase E). Ba,,ed on this data a model for the lot)ping of the B fragment through the membrane was proposed, in this model the previously defined hydrophobic strctches ~see section II) were envisioned as being transmembraneous. The region from 265-29.5 was proposed to loop through the membrane twice, either inserting fully or part way in order to accommodate the existence of flanking external digestion sites. In addition, the relatively hydrophilic regions from residues 2(X)-215 and 386-402 wele also proposed to be transmembrancous. This was necessary to ac,'ommodate the observations that the A-B disulfide becomes exposed to the cytoplasm upon insertion (see section XV). and th0t no

~A digestion ,wcurrcd wilhin fL" hydrophilic ('-terminal portion of the B domain implyh~g .~ cytoplasmic I.~calion for it An alternate possibility to explain the latter ~bservatior in that the C-terminal legion is folded in s,'ch a way that digestion sites ;ire not exposed. AIIhLugh it would be unusual if residues 200-215 and 386-402 were tr:msmcmbrancous based on what in kno~ ~ about other membrane proteins, it is quite possih~ • that the toxin will not fall into the oversimplified cla,,, ical picture of membrane proteins (see section VI). The tol,ography and confi~rmation of the membrane-insert 'd B domain was studied by the expression of toxin m~.'ants with an in vitro tran.~ription/ translation syst "m [69.115]. It wa~, fimnd that even after deletion of part or all of tile A domain the truncated toxins were ablc to bind to plasma mcmblancs and iq.',¢!;. ~:! ],~w pH ~,ilh a tt,l~;graphy similar to that of whole toxin as judge.I from the products of digestion by extcrn::liy added pror, ~se [115]. As judged from the ptt dependence of digcstit n. the conformation and insertioli of ihe truncated tox'ns exhibited the same dcpcndcncc upon low pH set.3 with native whole toxin. However, the conformation.'l changes occurred several tcnth'~ of a pl-t unit higher h'an lot native toxin (115]. The truncated molecules also ~.xhil-itcd pore pro=, :riles ~onsistcnt with their confi,rn itional and insertion properties. "I'hcsc studies are dis-usscd in section IX. smallci truncation mutant mis~;ng both the A domain and the N-terminal region ol the B domain did not seem to bind cells as well as mole "ules containing a full icngtil B domain. Topographical infi~rmation derived .rom studies of toxin pore formation is discussed in sect on VII.

VI. Why low pi= alters diphtheria toxin folding and hydrophobicity Protonation of amino acid residues is cen.ral to the changes in toxin behavior lit low p|{ The it entity of the residues :hat protonatc at Iov. pH combi= ed path the: crystal structure of the toxin would go far owards I:iving us the mcchan!sm behind the low p|-I i 'duccd ch::.".g':~. Xt present, wc c a n only outline the t~ ~cs ol residues and interactions likel~ to be involve,: ()f cot,.-,e, protonation of the carboxyl groups of Asl and Gla must be imlx~rtant. However, as ix~intcd out p :viously [34,57.86] His protonation is a l ~ very likcb to play an important role. despite the fact that the par al unfolding of the toxin occurs at a pH below the pK~ ,f a free His imidazolc ring. A role for His is l i k e " because the pK;, of a ills residue involved in act( denaturation will he Io~er in a Ioldcd protein than ir an unfolded protein, where it should be close to that o[ the free amino aci6. This is true because by dcfiniti,m acid-induced unfolding re0~irc., that the residues in-

volved bind protons more strongly in the unfolded coniormation 'ban in the folded conformation [57,110]. This is how proton binding shifts the equilibrium towards the unfolded conformation. Changes in tt;xin behavior at low pH include partial unfolding and increas,:d hydrophobicity. Wc will consider how protonation could induce partial unfolding first. One possibility would arise from the presence of unprotonatcd Hi: residues buried within the hydrophobic core of the pro',ein. (A buried unprotonated Lys or Arg could al,,~ bc involved, although unlikely.) Ulxm protonation an energetically unfavorable i~flated buried charge would form. This unfavorable situation would be alleviated by unfolding. A second I'mssibility would arise from a His r~.:siduc placed in a cationic environment in the folded protein. Protonation would give rise to cleetrostatically repulsions which again could be alleviated by unfi~lding. The loss of negative charge upon protonation of Asp and Glu ;csidues could also allow electrostatic repulsions between cationic residues to predominate at low pH. again promoting unfolding. Protonation of Asp and Glu residues could also result in unfolding due to the loss of internal salt bridges that stabilize the native state. The observation that addition of salt appreciably stabilizes the unfolded low pH fi~rm of the toxin [91l) supports the idea that these electrostatic changes control unfolding of the toxin at low pH. Salt can shield like ch::rgcs from one another, and since the ability of sail to penetrate between charges is greatest in an unfolded protein this can give rise to net stabilization of the unfolded state (even though electrostatic repulsions may he greater in the folded state)[I 17]. Electrostatic shielding by salt could also promote unfolding by destabilization of salt bridges found in the native state. The sequence of the toxin also contains clue,,: to the nature of the changes at low p g . The C-terminal region of the B domain is relativcl!,, talk,nit and rich in His residues. Therefore, electrostatic repulsions in this =-cgion may play a role at Iov, pH. Another factor may be the charged residues between the hydrophobic stretches of the B domain, which are mainly Asp and (;hi (57]. At low pH the protonalion of these residues could directly mcrea.~ hydrophobicity (see below) or ,nigh: break salt bridges to the cationic C-terminal domain. The various electrostatic factors that could influence toxin at low pH arc summarized in Fig. 4. The composition of charged residues in the A and B domains show an interesting contrast :hat may infl,'.once behavior at low pH. There is only one His in the catalytic A domain, but 15 His in the B domain. Therefore. protonation of Asp and Glu residues is likely to dominate the rcslxmsc of the A domain to low pH [85]. It is interesting that a similar bias is seen in the sequence of Pseudomonas exotoxin A [60] (.see section XX). It ,amuld a l ~ be noted that bias for Lys

35

l lOW pH 1

Fig. 4. Illuslralion ol Ihc ch:clro,,latic inl,:raclion,, likely, to bc altered at low pit. T h e toxin is represented a~, a flddcd pro=c=. ~,ith a domain h~mndau, to~.'ard~ the middle of the molecule. In the top slltl~.Lur¢, representing Ih¢ '~ilualion al neutral pll. the charges rcprc.,.cn! Arg, I,ys. A,,p and (Hu residue,,. It rcprc,,cnl, I li,, rc,,iduc,,, and Ih¢ slriped region rcprc,,cnt,, the hydrophohic ct~rc *.d Ih¢ protein. At Io~. pit the Jib. residues ;ire nov, ,,hov.n I~y + ",ymbol', and Asp and (ilo residue', h~. o symb~fi,, to md~c,.tc theft prohmalion. The clcctro,.taltc change,, ,~,ill I~.,. | I | prohmalion ol r.",iduc,, leading to charge rcpub, itm,, 1ha! dcMabiliz¢ lilt: hdding ~.1" a dolu.m~ or breakdov.n ot illtcrdomaill interactions.. 12) Io',,, ol ,qal)ih/ing ,~;lll hridg¢~, duc to protonalion ol anionic rc',itlu¢', ~.ilh Ih¢ xan|c ¢on,,cqucncc,,. (3) ~.in'~ilar change,, a-, Iho,,c mcnlitmc:l a|'~',¢ cau,,ing the ol~ning ol it hinge region, and 141 a dlrccl ;nch.,tsc in Ihc ~,iz¢ ol Ihc hydrophobic region or it.,. dcgrcc ol h~drophohicity dm. hi ]o~, ol ch;Hgc on ...Ii~.~llic rc',iduc',

vs. Arg usage, or vice versa, is seen within the catalytic domain of diphlhcria toxin, cxotoxin A and a number of other toxins [118]. Although the reasons for these biases are not known, one possibility related to membrane translocation is that the type of charged residues pre.~nt control the ease or reversibility of unfolding of the catalytic domain during translocation. Because of the lower pK,, and lesser hydrogen bonding of Lys relative to Arg [119,120] such a hypothesis would predict that toxins with Lys bias could take on an unfolded form in memblanes more easily during translocauon. One other residue that has been proposed to play a role in conformation changes at low ptt is proline

[121,122]. It has been pointed out that the rate of proline cis-tran~ isomeri;,ation is pit dependent [123]. and that at least one of tile hydr~+pho|,ie segments in tile h+xin ha,, a nt,mber t'l+prolines [121]. it should he noted that the equilibrium between cis and t r a n s proline is not pll dcpcndenl, since an kmization change is not involved, and that the energetic difference between a ci.+ and trans proline is not large [124]. "l'herel.+rc, interactions between other residues that are altered by prolinc ('is-trans isomerization would have to be inyoked to explain a large proline isomerization effect on the energetics of the confi)rmational change at k)w pH. The second aspect of low pH effects on the toxin is the induction of its hydrophobic behavior. Is it due mainly to a direct increase in hydrophobicity upon Asp and Glu protonation, or to the eXl~sure of buried hydrophobic mites upon partial unflHding? The induction of toxin hydmphobicity by unfl)lding in SDS, high temperature, and high pH suggests that unfl)lding rather than carhoxyl protonation (~,hieh should only occur at low pH) is the dominant factor. However. it is likely that c;~rboxyl protonation also play~ a major role in hydrophobicity. One reason is that the toxin appears to be more hydrophobic at low pH than aft:r thermal denaturatkm at pH 7 or after unfldding at high pH [9~.113]. In addition, as noted above the sequcncc of tile toxin shows that a number of uncharged scquc,~ces are broken by Asp and Glu residues [57]. This suggests that their protonation could greatly augment hydrophobicity. "I'I,~=~t,lc of carboxyl groups is clearzr for the hydrophohicity of the isolated A fragment. The A fragment becomes hydrophobic at Iov pH [56,g5], but not under unf't~lding conditions that do not involve carl~xyl proh;iiation, namely after thermal denaturation at pH 7 or unfolding at high pt! [56,113]. It has been propo.,:cd that protonation of th,' ,:arl~)xyl groups of Asp and Glu at h)w pH could allow A fragments to fi)rm oligomcrs that shield their polar groups from contact wi!h lipid and thereby aid membrane penetration [85]. Another question al~aut hydrophobicity is identification of the sequences giving rise to hydrophobic behavior. [:rom ihc sequence of the B domain we have seen that the existence of several transmembrane helices is likely. Itowever, the A domain shows hydrophobic behavior without having any hydrophobie sequences. One possibility is th~,t g~me hydrophobic sequences have been overkx~ked because the commonly sed hydrophobicity scales [125] are used without aCjustment for the pH dependent change in the hydrophobicity of ionizable amino acids. However, we have recently developed an adjusted hydropathy scale and find there ar~. :rely relatively small el=cots of pH upon hydrophobbci~,, of toxin scquer.ccs (unpublished observatZms}. ,,.aoLiler p~ssnbility is that other s:ruetures are involved. First, there may bc transmembrane {3-sheets.

36 The recent solution of the structures of bacterial l',t~rins have pr(wcd the existence of such structures in transmembrane proteins [126,127]. Unfortunatcl~. such /3sheets are extremely difficult to identify from ~J sequence analysis duc to their short length and mixcd hydrophobic/hydrophilic content [ 128]. Another I~*ssibiiity is that hydrophobicity ari.~s at the level of tertiary structure from a cluster of hydmphobic rcsiducs that are non--contiguous in the sequence [56,129]. This is consistent with the increase.d hydrophobic behavior observed for other partially unfolded proteins thai form molten globules. Finally, it should he kept in mind that there may be hydrophilic transmcmbranc sequences. ]f the toxin forms oligomcrs it has been pointed out that the number of transmcmbranc segments needed to contact lipid would decrease [86]. The possibility of .some degree of energetically unfaw)rable contacts between hydrophilic parts of the protein and the lipid a l ~ cannot be ruled out, especially because therc is evidence that upon insertion the toxin destabilizes bilaycrs ( ~ e section Xll). P-olinc i~)merization may al~) directly influence hydrophobicity. It has been reported that energy calculations for residues 423-440 are consistent with increa:~cd hydrophobicity if isomcfization trom ira,., to cis proline ()(:curs as pH decreases from 7 to 4 [122]. However, it should be noted that there is no direct evidence as yet for any change of prolinc i,~)mcrization within the toxin at low pH. The results from silc-(lircctcd mutation at these prolincs should hc very useful inl this regard [66,1~l].

VII. Pore formation by diphtheria toxin: bilayer lipid membrane studies The formation of pores in membranes by diphtheria toxin is one of its most well studied properties. In fact. the first demonstration that the toxin could End and insert into model membranes at low pH came from pore formation. The studies used to characterize pore formation will be di~u.s,~d in their (roughly) chronological sequence of bilayer lipid membrane, m~lel membrane vesicle and cellular systems. Two groups demonstrated that the toxin would form pores in membranes at low pH by measuring toxin effects on bilayer lipid membranc (BLM) ion coMuctance [131,132]. In Ref. 131 the C-terminal truncated B fragment (845) prepared from the CRM 45 mutant was found to form pores at low pH. It was also found that maximal channel conductance occurred when the c/s side of the membrane (the side of the m,:mbrane from which the toxin entered) ~,as at low pH 3nd the trmzs side was at neutral pH. This would be the situation encountered by a toxin entering the membrane from the lumen of the endosome ~,ad suggests thai there is oriented insertion of the toxin in the metrbrane. Pore

c o n d u c t a n c e was also dependent on membrane poten-

tial. requiring a c i s positive voltage. A ruughly sq,~arc dependence of conductance upon protein concentralion was found, suggesting that a dimer might be the conductive species. To assess pore size the passage of molecules of various sizes across multilamellar vesicles (MLV) bilaycrs containing inserted 1545 was measured at low pH via osmotic swelling. It was found that medium sized polyethylene glycol could pass through the Ix~re, suggesting that it would be large enough (18 ,A) to allow an unfolded A fragment to pass through. Pore conductance did not have a voltage requirement in MLV. In Ref. 132 the conductance increase induced by whole toxin was examined, l:k)th binding and pore form:ilion increased markedly below pH 5. Pore formation was favored by a ('is negative membrane potential althou[zh conductance levels were greater at a ('is positive voltage. No l~)rcs were formed by fragment A. It was concluded that the channel was small (5 ,A) based on the level of conductance, and that it might not be large enough to let the A fragment pass through. Again, the concentration1 dependence of pore formalion suggested that an oligomcr might be the conductive species. In Refs. 133 and 134 the conductance and opening/ cl,.,sing kinetic's of channels formed by CRM 45 and the B45 fragment were examined in more detail. Single channels were found to be either open or closed. Two types )f closing event were characterized, oboe type of which dosed the clnannel fl~r only a short time (flicker. ing) [132.134]. It wa,~ also found thai a large increase in transmcmbranc voltage decreased conductance. This was attributed !o an increase in the rate of closing events. The higher conductance found with neutral Inm.~ pH was attributed to faster opening events. Very similar c o n d u c t a n c e behavior was found for CRM 45 and B45. T h e lack of cffcct of the additional A domain in CRM 45 was consistent with channel fi)rmation occurring after the A fragment translocated rather than before. A wrapper mMcl was proposed in which the toxin inserted into membranes with the A fragment surrounded by the B fragment [134] (Fig. 2 (top)). Studies in which the conductance of CNBr fragments of the B fragment were measured determined tlnat only the CBI fragment, which contains many of the hydrophobic re~ions within the B domain (,see section II!), could induce pore formation [121.135]. In addition, CBI conductance was greater at low pH than at neutral pH. This supported its having a role in membrane insertion and pore formation at low pH. It should be noted that such experiments must be approached cautiously' because fragmentation could affect the. folding of a sequence in an unpredictable manner, t;specially as the Met-339 fragmentation site that borders CBI falls in the center of one uncharged

37 ~gment, and another hydrophobic stretch is divided in two by Met-314. In Rcf. 136 it was found that at low pH botulinum and tetanus toxins torm pores similar to those of diphtheria toxin. In this study it was also found that pH could alter the relative permeabdity of cations and anions through the diphtheria toxin pore, such that anion specificity at low pH switched to cation specificity at very low pH. it was noted that this behavior makes it difficult to determine pore size from conductivity experiments, it was also found that molecules as large as NAD + could permeate through the pore, ~pporting a relatively large pore size. Topographical information also has been obtained from studies of bilaycr lipid membranes (BLM)-inserted toxin. Inositol hexaphosphate (IHP) placed on the t r a n s side of a BLM (the side opposite that to which toxin was added) increased the conductivity of the toxin pore [137,138]. This occurred both at symmetrical low pH and when the t r a n s solution pH was neutral. 1HP effects could I)c blocked when ApUp was added to the t r a n s side. Since IHP is a P-site ligand and ApUp binding is believed to overlap the NAD* binding and P-sites it appears that upon insertion these sites reach the tratzs side of the membrane, equivalent to the cytoplasmic face. VIII. Pore formation: vesicle studies Several studies have used rm)del membrane vesicles to examine toxin pore formation. As noted above it was found that 1545 could make pores in MLV as well as in BLM [131]. in thc.~ experiments toxin was incorporated in the various shells of MLV and permeability of various molecules was asses~d by the rate of osmotic shrinking of the vesicles as~yed by changes in light ~attering [139]. Using the same ashy these results were extended using whole toxht [84]. From the dependence of osmotic swelling on the size of the permeant species the size of the pore formed by the toxin was found to bc larger titan that formed by bacterial outer membrane porins. A pore diameter of 24 ,~ was estimated. Also noted was that adding a reducing agent or using nicked toxin gave somewhat increased permeability. Permeability was also greater after toxin inserted at low pH was returned to neutral pH and went up quadratically with concentration, again suggesting pore formation by a dimer. in Ref. 140 the behavior of toxin in vesicles was compared ,o that previously found in BLM. The system used was LUV to which toxin was added after establishment of a membrane potential using a K + gradient and valinomycin. Pore formation was monitored by membrane depolarization, which in turn was assessed from changes in ANS fluorescence. Pore formation occurred only at low pH, with a transition to a highly

conductive state at pH 5.2. As in BI.M it was found that a ¢is positive voltage gave maximal p~re permeability. A linear dependence of permeability upon toxin concentration was also found, contrasting with the resuits of earlier studies. Several possible explanations of this were given, including: (I) the limited ability to follow initial permeability rates by fluore~ence: (2) a difference in the behavior of the first channels formed, which should be responsible for depolarization, compared to that of total or average channels; and ~3) oligomerization of the toxin prior to insertion. It should be noted that in this and the following reports the question of pore formation is complicated by the possibility of a transient permeability increase occurring at the time of toxin addition as a eonmqucnce of toxin-induced membrane destabilization or fusion. (A study of the relationship of permeability and fusion is de~ribed in section Xil.) Two factors mitigate the possibility of artifacts due to this phenomenon, First, the permeability propcrlies nf the LUV are similar to tho~ of BLM and MI.V containing inserted toxin, in which persistent permeability increases were observed. Second, transient permeability could play an important role in toxin trans!ocation (see ~ction XII). Nevertheless, it should be kept in mind that different studies ma~, be cxamining different aspects of toxin-induced membrane permeability. In Ref. 141 the release of entrapped molecules from LUV by externally added Ioxin was investigated. The pH dependence of vesicle entrapped caleein dye release showed a correlation with the pH of B domain insertion. At pH 5 the extent a n d / o r rate of calcein release was temperature dependent both iu vesicles containing dimyristoyl PC and soybean phospholipids. In the former case the lipid thermal phase transition could be involved, but the latter case might involve the temperature dependence of low pH-induced conformationaI changes (see section V). Release was linearly dependent on toxin concentration, and this was explained as in Ref. 140 (see above). One especially interesting result was that release of molecules of different .,,ize demonstrated that toxin induced lesion size was pH dependent, with larger molecules leakir$ out at pH 4 than at pH 5. At pH 4 even trapped 79 kDa dextran began to be released. The pH dependence of 'pore" size correlated with the pH dependence of insertion of the A domain, suggesting that the 'pore' could have a structure dependent upon exact solution conditions. in a second study vesicle permeability induced by insertion of isolated fragment A was studied [83]. Low pH induced relea~ of calcein that was roughly halfmaximal at pH 4.5 was observed. The fluorescence of the residual trapped calcein was interpreted in terms of a partial release from a vesicle, whereas whole toxin behavior fitted all-or-none release. As with whole toxin,

3X

"pore" size appeared to bc larger as pH decreased. These results, are surprising in the context of early models that envisi(mcd the A domain as a hydrophilic protein passing through a pore fi)rmed by the B domain, hut les,, st) in view of the data showirg that the A domain d¢~,'s insert in membranes lit h)w p l l (see sections IV and V). Earlier sludie,, may have failed to see (ragmcnt A induced "rs~res' because of the hr,vcr pH needed to inducc fragment A insertion. The requirement for very low pH probably means the pore t~)r,'ning properties of fragment A do not play a role under physiological condi,iom, (see .-,cotton XVII). Pore formation can a l ~ be detected when toxin is entrapped in vc,,iclcs and then exposed to low pH. This phenomenon i, nd its rclalionship to transh)-'ation is discussed in :.eolian X V I I . IX. Pore fi, rmation in cells

Several studies have examined toxin-induced I~re formation in Vcro cells. In Rcf. 142 it was found that after exposure of plasma membrane bound toxin to low pH the toxin fl)rmed a cation-specific leak thal allowed rapid influx of monovalenl cations, including protons. N a ' . choline and glue¢lsamine. Cation pcrmcahihty was bk~ckcd hy ( ' d : ' . There ~as little or no toxin induced anion permeability. Cation permeability was ab,o characterized in Rcf. I(h'~. Increased Na " influx and K" elflux was found at pH 5.5 and bclnw, corresponding to the pt! necessary fi)r membrane insertion. In general, the concentration (;f toxin necessary to induce ion translocation was orders of magnitude higher than that necessary tar protein synthesis inhibition. This presumahl.~ aro,,e from the r.cccssity of having many open pores within the plasma membrane in order to obtain i, detectable leak. When mutant toxins or cell lines were used in which higher tax;, ,.t,.~.~=,,.atiom, w~.-,= n,.cdcd to induce loxicily lhcre was a corresponding increase in the concentration needed to induce l,crtncahility. It was al~) Iound that the incubation of c~:lls with an excess of toxin led to a preferential enhancement ol permeability relative to cytotoxicit~, suggesting that toxin oligomer~, reducing htrger or marc numcrou,, pores might fi~rm lit high toxin concentrations [1(~]. When pH was reversed to neutral the ion Iluxcs ~/crc inhibited. ['hcrefore. it was proposed that toxin induced pores closed at neutral pH. However, this is complicated by effects due to the pH dependence of cellular transporters that would counteract toxin-induced ion flux. One would predict these pumps are much more active near pH 7. In Ref. 142. inhibitors were u,~d to shut off cellular pumps. In that case residual permeability was observed upon reversing pH. However. it should be noted that the efficiency and pH

dcpendencc of inhibition might complicate quantilati~,e interpretation. From the leakage of various small molecules it was inferred that the size of the pore fl~rmed in cells was small [143]. Increased cffhix was observed fl~r cations and aminob,ohutyratc hut not tier presumably phosphoryll:tcd deoxyglucose. In this study, it was a!so reported that ('d ~' could inhibit Icakagc in mu._l~.l metal,lanes as well as in cells. 1"he effects of a series of truncated toxins on pcrmeabiiiiy wc-e studied in Ref. 144. It was found that the B3h deletion mutant (essentially equivalent to the B fragment, see section Ill ) could induce .r,w¢ fi~rmation m Vero cells. l h c s c pores had the same spccil'icity as those formed by whole toxin, i)ore formation by B~h ~;:,, even more cflicient than that of native toxin, requiring two orders of magnitude Ics~, concentration. The pH dependence of pore formation aml conformalion (~cc ,,cotton %') of B3h were similar to that fl)r re,live toxin except ~hiltcd upwards by a few tenths of a pit unit. "l'hc,,e rcsuhs were consistent ~ith a ml~lel in which pore fc~rmation was due to the t3 domain, and in ~hich the absence of the A domain hath de~,tabilized the i] domain towards low pH induced partial unfolding ,,l~d "unplugged" the ix,re, l)elclion mutants missing the N-terminal ic:i..,ion of lhc B domain also formed pores hut they were neither specific nor sho~ed pH dependence. X. The effect of lipid structure on interaction with diphtheria toxin and the origin of electrostatic effects on interaction

l'hc interaction of toxin v, itl= different types of phospholipids was fir,,t examined i l Rcf. 145. It was found that MI.V coml,~sed of only zv~itterionic p~o,,pholipid,, v, ould not hind ;i,, ,,trongly u. 1115 toxin as vesicles, containing anionic (negative) lipd~,. 1"his was attributed to a toxin-phosphate intcracti m dependent on the degree of phosphate exposure Ii;!her than to an elee;rostatic +'.lcct. It wa,, also claimed that comFs)unds such a ;;h~+:Fhccht+line could act as it,;fibitors of toxin binding It+ liposomc~,. Unfortunately. this study was performed just ncfl~rc the realization that pH controlk.d toxin hydrophobicity. Many experiments were done in the pH 5 - 6 range in apparently unhuffcred media. This pH is just at the Ix~rderline of the toxin's hydrophilie-to-hydrophobie transition. Therefore, some results may have reflected lipid binding of toxin in its low pH conformatkm and others lipid binding by the native state. Interestingb. in one experiment it was shown that toxin b~mnd best to ~ m e membranes at low pH. Several subsequent reports were consistent with a stronger interaction of toxin at low pH with model membranes eontaLting some anionic lipids than that

39 with zv, ittcrionic ones. First, I~re formation ~as shown to bc greatly favorcd in anionic membrancs[ 131.14(I]. Second, toxin-induced membrane aggregation and fusion was much greater with ankmic vesicles [14~]. And third, the degree of toxin photolabcling by hydrophobic agents was greatest with anionic vesicles [85]. These findings were attributed to electrostatic interactions. "v'cly ~ecemly. preferential interaction of toxin with anionic lipids was observed using monolayers [i113]. In Rcf. 147 the details of the effects of lipid structure on interacti*m with toxin were iu~,cstigated. Tight interaction of toxin with lipid vesicles was found only at low pH. Toxin associated with SUV regardless of charge, as expected for a basically hydrophobic interaclion, but as.,a)ciated more strong!y with vesicles containing ,~)me (20":,;) anionic lipids than with zwitterionic ones. The difference between binding to zwittcrionic and negatively ehar;,,:d :c:Jclcs was evcn greater if LUV were used or if toxin was preincubated at low pH before addition of vesicles. No inhibition of bind!ng by phosphocholine, ApUp ~r IHP was found at low pH. This supports the conclusion that electrostatic factors rather than phosphate interactions arc rest)risible for the stronger interaction of toxin with vesicles containing negative lipids at low pH. This was confirmed by the observation that the pH dependence of toxin binding to vesicles depended on lipid charge but not on the chemical identity of the lipids. In the pre.~nee of 20c; anionic lipids there was an upwards shift of 11.2 unit in the pH at which lipid binding ix:cuffed. One likely factor in this shift is that the It~cal low pH that surrounds anionic vesicles would cause the toxin colliding with a vesicle to undergo the hydrophilic-to-hydrophobic transition even when the bulk ,~)lution pH was tt~) high to induce the transition. Similar conclusions were reached in a study of the interaction of tetanus toxin with different lipids [148]. in addition to local pH effects there arc several factors that may induce strong binding of toxin to anionic lipids. One factor ix th.Jt at low pH the toxin can its a Irx~sitivccharge [147]. Furthermore. at low pH the partly unfolded hydrophobic form of the toxin must be more protonated and therefore more posit=rely charged than the native confornmtion at the slsme pH (see section VI). For this rca,~m, electrostatic interactions should bc most favorable between the hydrt)phobic form of the toxin and aniouie vesicles. Another lactor could be the effect of toxin insertion on electrostatic repulsion.,; between anionic lipids. A decrease in repulsions between anionic liplds cotdd tK:cur upon toxin insertion because insertion should increase the average di~t;:nce b¢iwccn .,he negative charges carried by the lipids. Thi,~ should provide ;m extra driving force for insertion. Interestingly, as in the ease of SDS-protein interactions, such an effect would not be dependent on protein charge.

In this context, it is interesting lhal loXm interaction with negative lipids sho~s striking parallels Io protein in',eraction with SDS [12q]. in both cases protein interaction with amphipathic anionic molecules ix involved. In addition, both SDS and lipid interaction involve binding to partly unfolded protein, and I~)lh can involve hydrophobic interactions despite the absence of hydrophobic sequences in the protein [56,129]. Therefore, the interaction of some proteins with anionic lipids may bc more similar to SDS-protein interaction than generally realized. Differences between interaction with anionic and z~'itterionic ~,esicles have also been observed fi)r i~)lated fragment A [50.85]. For fragment A the upwards shift of insertion pH upon addition of anionic vesiclcs relative to Ihat with zwitterionic vesicles was grcalcr than the shift observed for whole toxin. This suggested that fragment A interaction wits especially sensitive to electrostatic influences, although subtle dificrcnces in vesicle sizc ma.v a l ~ bc a factor in this behava~r [56].

Xi. Interaction of native diphtheria toxin with lipids at neutral pH The analysi.~ of toxin imeraction with lipids at neuhal pt! h':s been complicated by the fact that the interaction ix much weaker th~,n at low pH. However, thcrc appears to be some significant binding of native toxin to lipid model membranes at pH 7. In an curly stud,,' toxin at neutral pH wax found to associate with model membrane vesicles at extremely high lipid concentrations [149]. Toxin-lipid interaction at pH 7 was al.,~) obse~'ed in an IR analysis [35]. Generally, very weak labeling of toxin has been found using membrane-inserted photolabels at pH 7 [82,85]. In one study, a photolabeling group placed in a shallow membrane location gave more labeling than a deeply placed photolabel, suggesting an interaction or collision o| the toxin with the vesicle surface [St)j. There has bctn some evidence for a specific interaction of toxin with PIP via the P-site at neutral ptl [145]. Based on the possibility of specific interaction, it was speculated that lipids like PIP could play a re~:cptm function for the toxin. However, it has been found that PIP and tee P-site ligand IHP only affect toxin pore formation when present on the t r a n s side of BLM [137]. This result argue,,; against a receptor function because it involves lipid on what is equivalent to the cytoplasmic side of the membrane (see section VII). In addition, the evidence that there is a proteinaceous receptor for the toxin (see section XVIll) makes it unlikely that lipid acts as a receptor under physiological conditions. Nevertheless, it is possible that lipids could act to supplement receptor binding [150,1511. it has been pointed out that a dual interaction :~f toxin with lipid

40

and proteinaceous receptor would result in a stronger total binding constant cqual to the product of the individual binding constants for lipid and receptor [ ]50]. This model was developed to explain the hindir~ .1~,. havior o f CRM 197. which displays hydrophobic behavior at neutral pH (sec section XVI). We do not yct know whether receptor-bound native to~in is positioned :,,) that it can simultaneously bind to the lipid bilayer. Xil. Toxin induced membrane fusion and aggregation Several studies have shown that diphtheria toxin can induce membrane fusion at low pH. In Rcf. 152, fusion of PC SUV vesicles by toxin was demonstrated. Low pH was nccessar/for fusion. This fusion event involved both the mixing ol lipid molccu!cs from two vesicles, as determined both by DSC and fluoreseence polarization. and mixing of their internal aqueous compmtment,~ as determined by the terbium-DPA ashy [153]. The investigators noted that since the N-terminal part of the hydrophobic CBI fragment could form an amphipathic helix thcre might be an analogy to the behavior of mclittin, an amphipathic helix forming peptidc that also has the ability to fuse membranes. in a second study [146] an NBD/rhodamine energy transfer assay was used to show that the toxin induced both aggregation and fusion. The total of fusion plus aggregation was greatest for membranes containing anionic lipids. Interestingly, both isolated A and B fragments could induce fusion, as well as the CBI fragment of B. It was also found that addition of ATP inhibited the fusion process. This was attributed to ATP binding to the P-site. However. at low pH a number of regions on the toxin should bc cationic and might interact with ATP. Aggregation and fusion of SUV were further studied by toxin-induced increases in vesicle turbidity [154]. Turbidity increa~s were low pH dependent, wi0h :~ midpoint at pH 5.8 at 45°C. Using an energy transfer assay at pH 4.5 it was demonstrated that the low pH induced turbidity change involved .some fusion as judged by lipid mixing. At pH 5, 45°C there was no leakage of calcein from the vesicles upon fusion. The dependence of fusion on the concentration of toxin suggested that a small oligomer of the toxin was ~ikely to be involved in the fusion event, The fusion of LUV by the toxin at 2(PC and its relationship to release of encapsulated materials was studied in Ref. 155. It was found that the leakage of calcein from membranes was tightly correlated with the insertion of toxin into membranes at low pH, showing the same lipid composition effects as found for toxin insertion [147]. This leakage could be induced by the toxin and its isolated B domain but not b:, the A domain, lnte~'cstingly, release could bc found under

conditions in which fusion had not occurred. Thcrefore, the investigators concluded that fusion and release were to .~)mc degree independent events. They attributed release to bilayer destabilization by the toxin. It is not clear what they considered to be the relationship, if any, between this destabilization and pore formation by thc toxin. A model for fusion was proposcd in which an electrostatic interaction between toxin and lipio, which would bring vesicles together, is followed by an insertion event which makes the vesicles more prone to fuse. The results of the tx,,o studies described above seem to disagree on the linkage of calcein leakage and fusion, ltowcver, the conditions used differed in lipid/ toxin ratio, temperature and vesicle size. Vesicle size, it should be noted, has a tremendous effect on the toxin to vesicle ratio. One important question these studies raise is whether toxin-induced fusion or membrane destabilization could play a role in toxin translocation. Although endosome-tr.':ppcd toxin should not induce intervcsiclar fusion, it could cause fusion between different parts of an cndosomal membrane. Alternately, toxin insertion could simply disrupt the membrane transiently and allow its own movement onto the cytoplasmic face of the membrane. Complete lysis of the cnck)some by trapped toxin seems unlikely. Even with many toxins per model membrane vesicle only small amounts of large molecules will leak out of the membranes (see sections Viii and XVil) There is evidence in cell culture that has been interpreted to indicate that a large number of toxin molecules burst into the cytoplasm at once, and that .~me ,~)rt of hilaycr destabilization is a likely mechanism far such b':havior [156,157]. The supporting evidence for this burst comes from a kinetic analysis of the intoxication process. The complex behavior of cells makes it difficult to extrapolate from such studies to the prcci.~ molecular behavior of the toxin. For example. local cell density on a plate might complicate irltcrpretation of kinetic cffects because it has been shown that the number and affinity of toxin receptors is a f , nction of cell density [158]. Nevertheless, transient changes in the membrane that are induced by in~rtion may well play a role in the translocation process. Xlll. Toxin aggregation and oligomer formation One of the most important unanswered questions about diphtheria toxin is whether toxin oligomer formation plays a role in its action. Although there is evidence that the introduction of a single A domain can eventuaX~:,'kill a cell, this process may take many hours or days [159], and at the other extreme it has been argued that : large number of toxin molecules

41 operate in a highly cooperative proccss [156,157] (sce .section X I I ) . Low pH induced toxin aggregation in solution has been examined in a number of studies [M,92] (see .section V). It is likely that much of this aggregation arises from contact between the hydrophobic sites on the toxin that would normally contact lipid. In this way the aggregation would substitute for the membrane, and would explain the similarity between toxin conformation in solution and in membranes at low pH [I 12]. Aggregation can affect toxin interaction with membranes. Allowing the toxin to preincubate at low pH in solution inhibits its subsequent binding to PC vesicles. However, in vesicles containing 20% PG this inhibition is totally abolished, suggesting that the aggregates arc dissociated by the lipid [147]. There is also evidence, mainly coming from studies of pore formation, that the toxin can form oligomers within membranes ( ~ c sections VII-IX). This oligomerization may be quite distinct from the aggreg;, tion in solution. Oligomers in a membrane would presumably have their hydrophobic face in contact ~,;th the lipid rather than each other. It has been poi~ited out that this type of oligomerization would reduce the amount of an individual toxin molecule that is in contact with the lipid, and thus allows efficient insertion where a monomer could not fo,'m a totally hydrophobic interface with lipid [861. Studies which show a square depenr~]ence of permeal-,ility upon toxin concentration suggest toxin forms a dimer (see sections VII and VIII). However, there are other possibilities, such as the dimerization of a non-conductive oligomer to form a conductive one of double the size or that there is a mixture of oligomers with sizes dependent on toxin concentration. We have found evidence of oligomcr formation in membranes using the selfquenching of rhodamine-labeled toxin and by chemical crosslinking. These oligomers appeared to be composed of tightly bound toxin molecules because they did not seem to break up at high lipid concentrations (D. Tortorella and E. London, unpublished observations). The obsetwation that the pH needed lor toxin to kill cells needs to be less extreme at high toxin concentration also has been proposed to be an indication of oligomer formation [86]. However, this effect would be expectcd in any case due to the usual increase in cytotoxicity with increasing toxin concentration. XIV. Cleavage of the link between the A and B domains The 'nicking' site between the A and B domains occurs at a cluster of basic amino acids. The toxin is easily cleaved at this site by trypsin in vitro [160]. The proteinase that attacks this site in vivo has not been determined. It has been shown that in vitro urokinase will nick the toxin between A and B domains, although

more slowly than trypsin [IOIL and it is possible that in vivo a urokinase-likc protcinasc nicks the toxin circulating in the plasma or at a cell surface. Since cndosomes contain proteina~s [162] it is also possible that nicking occurs within the endosome. In this regard it is very provocative that the toxin contains a rec('ntly determined consensus ~quence for vesicular ptoteolytic processing [163] at its nicking site. Another possibility is that the cleavage is opportunistic in the sense that it occurs at different times and places depend:ng on individual conditions. Such behavior has been ob.served for the nicking of the influenza virus hemagglutinin protein [164], which like the toxin undergoes a low pH-dependent hydr~)phobic transformation within endosomes [94,165]. The nicking of the toxin is probably necessary fi)r its function. Without nicking the A fragment could not he relea~d into the cytoplasm. It has been found in cell culture that nicked tcxin is able to inhibit protein synthesis more rapidly, and is more l~)tent than unnicked toxin [81]. Presumably, the unnicked toxin must wait for proteolytic processing before it can complete its translocation. Nicking does not seem to have any effect on conformational changes [34] or membrane in~rtion at tow pH [82]. However, the exact nicking site may affect insertion and olay an important role in translocation (see section XVII). XV. Role of diphtheria toxin disuh'lcle bonds in entry The reduction of the A-B disulfide appear~ to be necessary to rclca~ the A fragment into the cytosol and allow it to catalyze ADP-rilx)sylation [7]. Since the extraccllular medium is an oxidizing environment and the cytosol is a reducing environment one early suggestion was that fragment dissociation occurred after cytosolic exposure of the A-B disulfide and its reduction [7(I]. There arc several reasons to think this mechanism might be valid for toxin penetration through the endosomal membrane. First, endosomal contents are probably derived from the extraccllular medium, and therefi)re the endcsome lumen is likely to constitute an oxidizing environment, although the possibility of transport of a cytosolic reducing agent such as glutathione into endosomes has not been eliminated. More important, ',here is ran;sire inhibition of reduction by sulfhydryl reagee,:.~ such as glutathione in the low pH environment of the endosomal lumen. The reason is that reduction of disulfides by sulfhydryl reagents involves attack by the ionized S- form, and since sulfhydryl pK~ i~ near 10 the concentration of S- at pH .5 will be 100-fold less than at pH 7 [166]. Although there is little direct information on disulfide status in the endosomal lumen, it is interesting that disulfide linked cystine can accumulate in lysosomes [167], and reduction of endocytosed probes seems to occur in the

42 (iolgi rather than cndosomcs [168], suggesting that disulfide reduction is indeed inhibited in acidic vacuoles. In vitro properties of the toxin arc consistent ~ith this behavior. It has been shown that low pH treatment of m,3dcl membrane-in,erred toxin followed by pH neutralization and the reduction of the A-B d~sulfidc induces the release of the A fr;~gmcnt into .,a)lution [112,177]. ,~n cells, reduction and release of fragmcnt A into the cytoplasm has a l ~ been shown to occur after exposure of surface bound toxin to low external pH [169]. These observations are di~ussed more fully in the context of translocation (see section XVII). A very different process of disulfide behavior was proposed in one study [171p]. It ~as found that the disulfides of the toxin had to bc initially intact for efficient t~;xicity. A model was proposed in which disulfide c~,change that transferred Iragment A from linkage to the B t;agment to a series of proteinaccous membrane acccptors was important fl~r translocation. There is no evidence for existence of these hypothetical acceptor molecules. Hogevcr, it should bc pointed out that protein participation in disulfide reduction cannot be ruled t~ut at this time in view of the observations that thiorcdoxin can efficiently reduce the A-B disulfide, even at low pH [169] (see section XVII) There is e~,,en less information about the role and behavior of the disulfide bond within the B domain. Exl~erimcnts with the B disulfide-lacking mutant CRM I(101 indicate th;,t although it is able to penetrate membranes similarly to wild type toxin the B disulfide must be intact for efficient toxicity, perhaps due to ~)mc requirement at the translt~:ation step [86] ('.~cc section XVI). On the other hand, other mutants in which B domain ~steines are removed show a normal pattern of pronase digestion upon binding and penetration of the plasma membrane [I 14]. XVI. Membrane interaction and con~'o~-malion of natural mutants Gf diphtheria to~3n

Naturai mutants of diphtheria toxin, cal!c:~ CRMs (c~'oss reacting materials), have been very useful for investigation of toxin structure and function. CRM 45 is probably the most well studied CRM. It is m;s,ing the C-terminal half of the B-domain, terminating at Thr-386 [171]. Experimentally, the fact that the fragment of the B domain derived from CRM 45 (c:~!!ed 15451 is water mluble has made it useful for examining pore formation [1311. The hydrophobicity of CRM 45 was di~ussed in section IV and its pore forming properties in section VII. Additional studies have found that CRM 45 can bind to mtulel membranes [651 and insert into monolayers [il13] at neutral pH. It also undergoes a transition to a state showing increased insertion at I(wv pH [1(13]. This transition occurs at a

slightly higher pH than that in whole toxin. In contra~t. fusi,~, and aggregation of membranes containing anionic lipids by CRM 45 was found to require a lower pH than that by whole toxin [154]. This seem: puzzling at first because fusion is promoted by the toxin in its hydn~phobic conformation (see .section XII). However, as noted in section XI! electrostatic interactions are also important for fusion and CRM 45 is missing the cationic C-terminal region [154]. An electrostatic effect is consistent with the observation that at pH 4.5 there was more fusion and aggregation of anionic vesicles caused by whole toxin than by CRM 45 but there was no difference with zwitterio~ic vesicles [154]. CRM 197 is another mutant that has been studied in some detail. In this mutant Gly-52 in the A domain is replaced by glutamic acid [171]. Using soybean lipid SUV containing photolabels, CRM 197 gave increased insertion of both A and B fragments relative to wild type toxin at pH 7 [1511]. This was attributed to a changed toxin structure in the mutant at pH 7. Interestingly, the low nH induced transition to the highly hydrophobic state ~:ccurred at the same pH in CRM 197 as in wild type toxin. CRM 197 membrane insertion was also examined by photolabeling using LUV [i51], using different lipids than in Ref. 1511. In this study the most prominent difference between CRM 197 and wild type toxin at neutral pH was the increased degree of photolabeling of the A domain in CRM 197. In addition, the fluorescence and increased trypsin sensitivity of the CRM 197 at neutral pH was similar to that of wild t;,n':, toxin at low pH. "l'hcrch;rc, it ",~a:~:~uggc:~tcdCRM 197 takes on a mt~re insertion-competent conlk~rmation than wild type toxin at neutral pit. Nevertheless, there was still an increase of CRM 197 insertion at low pH comparable to that of wild type toxin, in agreement with [1511]. One way to explain these rt:~ults is that in CRM 197 the A domain may be already unfolded at neutral pH, hut that the major change in B domain structure occurs only at low pH. as usual. Unfolding of the A domain at pH 7 might allow insertion at pH 7 by exposing some hydrophobic sites on the B d,)main. ]'he fact that A tit)main labeling is pH-dependent in CRM 197 may reflect the fact that fragment A hydrophobicity is pHd~p~'n,.t.,'r .cvcn when i= is in thu unfolded state [56]. Another mutant that has been examined is CRM I(101. This molcculc contains a substitution of Tyr for (?ys at position 471 [154]. As a result the disulfide bond within the B domain is unable to form. it was found ~hat the pH-dependent insertion of CRM 1001 was very similar to tha! of wild type toxin [861. The only difference was that the t~,tnsition pH for the B domain was 0.2-(1.?, ,~ni!s lower than for wild tvr)e toxin. It was pointed out that th.s could be the cause of the markedly lower toxicity of CRM I1~)1 because it might prevent insertion at endosomal pH. However, it was also found

43 CRM I(X)I was several orders of magnitude less toxic , than wild type toxin after exposure of cells with bound toxin to pH 4.5 medium. Since both CRM 1001 alid wild type toxin are maximally inserted at this pH. it was concluded that the loss of the disulfide bond must alg~ affect a step in translocation subsequent to in.~rtion. It should be pointed out that somewhat different behavior was found for CRM i(Xll [173] as judged by partitioning in Triton X-II4 [172]. It was found that CRM 1001 underwent no distinct hydrophobic transition but was instead somewhat hydrophobic over a ",~hole range of pH, although not as hydrophobic as wild type toxin at low pH [173]. It is possit,',e that the iodination of the CRM 1001 u ~ d in this study perturbed its folding. Two other mutants that have been studied are CRM 9, which has a B domain mutation affecting receptor binding, and CRM 176. in which there is a substitution of Asp for Gly at ra~sition 128. These molecules exhibited the .same as hydrophobieity ploperties as wild type toxin [15(1.151].

XVll. Diphtheria toxin translocation across model and ~et.! membranes Although translocation mechanisms have been proIx)~d ba~d on various experiments, expcr=mcnts that directly assay translocation should be especially imps)rrant for defining the translocation mechanism in detail. In Ref. 174 the transkx:ation of diphtheria toxin across model membranes was assayed using the ADP-ribosylation of vesicle-entrapped EF-2 to detect tran:;!:'e:: lion of the A fragment into the vesicle lumen. In this study, EF-2 and N A D " were trapped in vesicles by freeze-thawing. Since thmsloeation was assayed via activity, an absolute quantitation of the amount of translocation was not possibh:. Nevertheless, it was possible to detect some translocatkm since ADP-ribosylation was obser~,ed when toxin was added externally to vesicles containing EF-2. To obta'n tr;m.,,i~.a|ion i: was necessary to treat toxin at low pH and add D'IT to reduce the A-B disulfide. Only a brief exls~sure to low pH was ,ecessary to induce ADP-ribosylatio, a~.iiviLy. consistent with the rapid conversion of the toxin to a hydrophobic conformation at low pH. One caution we should note is that in our hands u.~ of freeze-thawing to prepare entrapped protein has occasionally given rise to some protein associated with the outside of the vesicles. Translocation haz a!so hc.en examined using toxin efficiently entrap.r~.d in LUV [175] prepared by a desiccation/rehydration method [!76]. In this report lysozyme was co-entrapped with the toxin to aid trapping. Using crosslinking it was found that the entrapped toxin in~rted into the membranes at io*, pH.

Lactoperoxldase-catalyzed iodmation indicated that after a brief low p}t treatment some of the inserted toxin became eXl~scd to the external solution, and thu., the toxin had at least partially translocated. As a control Ior low pH induced changes that might increase lactoperoxidase labeling of a small amount of untrapped toxin attached to the outside of the vesicles it was shown that in the absence of lipid labeling of toxin in solutiot: was the same before and after low pH exposure. This control assumes toxin to irreversibly undergo the same low pH induced changes in the pre~nce and ab~nce of vesicles, as is found in most cases [I 12]. The details of translocation across model membranes have recently been investigated [177] using toxin entrapped in LUV prepared by octyl glucoside dialysis [178]. Entrapment was demonstrated by resistance both to protcolysis and to denaturation by guanidinium-CI, a membrane impermeable agent. Efficient translocation was ob.~rved after exposure to low pH followed by pH neutralization and addition ~f DTI', with rclea~ of half of all of the A fragment molecules. This was not a specific process because entrapped 17 kDa dextran was translocated as well. In addition, release was not conformation-specific, being equally efficient for membrane-inserted toxin containing folded and unfolded A domain [I 12]. These results are consistent with translocation by toxin-induced pore formation. However. deftnitc difference.~ i~, the transmembrane tolx~graphy of different conformation,~ were detected, with more of the toxin reaching the outside surface of the membrane after low pH CXl:X~sure when the A domain was unfolded. This concluskm was mainly h ~;cd on the de['r,'c (.)f complex fi)rmation betweer :,apr~ed biotinylated tc,xin and external streptavidin, it implies that confiJrmation-specific events play a role in translocalion. The distinctions between specific and no='t-specific pr, v,:esses are shown in Fig. 5. At present, wc have no definitive exphmation for why non-specific and specific, romponents of tr;:nslocalion co-exist. One possible cxplanatkm is that the vesiclc~ used each contained on the order of 200 toxin m,~lecules. In this situation even inefficient formation of a large lx~re could induce release as king as each "~esiclc has at least one I~)rc. At very low conccntralions of membrane-entrapped toxin a confiwmatkmspecific process might predominate. Alternately, if pores z:re formed by various toxin oligomers (see sectic,n 'Kill) then pore size might be concentration dep~.ndent and at low toxin concentration the pore might be too small to allow fragment A through. This would not be surprising as an inerea~ in pore size with increased oligomcr size has been found in the case of complement system [179]. In terms of in vivo relevance the question then becomes how many toxin molecules will an e n d o ~ m c contain, which is not yet certain (see sections Xli and Xiil). It is conceivable that even in

44

NO.~ - ~,P[C!~!C TRANStOCATION

½

_I-71

(D,.

SPECIFIC TRANStOCATION

/'~o'b.~ome /,men

~.~ I

(ytocr/tTt~m

i the t| ~.'It.~llll o| .I D.c,.Ill nlol~:t uh.' t l l l h plo1111~l~.'~ l h ¢ Ir~ifi~,hl~.'illlOll i~I II~ ov=R A t.hilHl..~rll.'t'lhc CIIlI]IHIII~I|IOII ~. g|,l~. |*=U rCqlllll.'d iI] lhi', lWOt:c', ", 111 nol1-~,l~.'ClllL" ll',lll~hlt.lhC, ll lh~.' II thilil1~ a l l o ~ ll-.in~,h~.'~lllOll ¢ii ..'~ ch;lil1', cl'ql~lllq~ |10111 o l h c r h~'~in iY~ol~'~.'ul~'~ iHltl, il dtlt.' Io I ~ i ¢ '*1,111,1,* . . . . . :*,.:: .! .~.:~' ';~ ~i I :l!!~I'l I~''!~ t."l!;.". ~ I.I~ =.t."li

~,ivo different mechanisms operate depending on the toxin concentration. One ~)mev,'hat surprising resuh I'r,m'= this study is lhat entrapped isolated A domain wa~ apparentl', able Io selt/tran~,locate, being released after cxpos,:rc t~J low pH and subsequent pit neutralization. This event occurred too efficiently to he readily expiained hy fragment B contamination This sclf-translocation is consistent with fragmcnl A properties at low pit .,,howing that it can insert and relea~,e ver,icle-cntrappcd molecules at Iov. pll [~3]. However. it raise,, quest,ms about the role of fragment B in fragment A transh~ca|ion. Wc fecl self-transh~calion of the A fragn~cnt b. an artifact for se~,,:ral rea.,,om,. First. isolated lragmcnt A becomes fully hydrophobic at a pit lower than I:1:11 fimnd in endosome~, 156]. Second. it has bccn lou,td that in a set ~i immunotoxins there is progressively less toxicity when the B domain is truncated or removed [180]. In addition, it is unlikely that the in vitro conditions of a large number of frec A fragments within a vesicle would ever be duplicated within the cndosomal lumen in vivo. Nevcr'hclcs.~, the tact that se!f-translocation nf the i.~flateu A Iragment can occur in vitro, coupled with the other studies documenting its membrane interaction, does suggest it plays an acti~,x, part in the membrane translocation step. One limitation of these vesicle studies ;:.; that pH gradients a=c unlikely to be maintained [ur more than a .

few ~ccon,.l,~ due to the p~rc formation by the toxin (even in objects a,, large as cells lhe toxin can destroy a p l l gradient in minutes [142]). Therefore. the behavior of the toxin ur~on exposure to the cytoplasm must be mimick'd by raising the pH on both sides of the membrane. This probably differs from physiological conditions, althemgh the possibility that the toxin could c, llapse the pH gradient across the endosomal n.cmbrane in ~ivo ha~, n,~t been eliminated. A potential problem when using biotinylated toxin may be that even though its properties appear to be quite like tho,~ of the unlabeled toxin [177], there may be structural changes due to biotinylation that are t(~ subtle to detect. Also. the likely presence of multiple biotinylation site,,, makes detailed topographical interpretation impo,,,,ible. Sterne investigators have concentrated on a cellular assay of transloealion using external acidificalion to help translocale the t¢~xin acr-~ss the plasma memhranc of Vero cells. This system has the ad~,antagc of h~king at toxin transhv,:ation across a nalura] memhrane, where cellular factors thai mity pla/ a role in transh~:ation, such as the receplor, will he present. In addition, in the cellular s.vstcm pit gradients across Ihc membrane can he maintained longer [142], and the effects of the reducing environment of the cytoplasm may he cvalu;dcd. In Ref. I1~1 it was found thai, in addition to a rc,quirement for low pH. a pH gra-licnt across the plasm;I n]emhrane was necessary for toxic ity and therefore Iransh~:alion. In this study the ,-3'loplasndc pl I. as well a~ extcrm.I ptl, was hrielly decreased. It was f, mnd that low cytoplasmic p l l was able to protect cells against the toxin, I~ut that if the external pH ,.~as at I~..asl I unit lower than cytoplasmic pH full toxicity was ohscrved. It is possihlc thai the pl-! dependence of pore size has a role in these effects, hecause mcmhrane conductance (and presumably l~)re size) is much greater -,ith a p l l gradient thilR with symmc'ric low p i l [131]. Another possihility is that ~he tendency of the membrane-inserted toxin to become more hydrophilic when exposed to higher ptt may result in its net movement to t~ne side of the membrane only when that side is at a significantly higher pit than the other. It is also I~ssible that Io~ cytoplasmic pH affects other cellular mL,h.'t.~ies that modulate toxin entt3'. This study also lomld lhal the absence of a transmembranc potenlial did not grcatl~ affect toxicity. However. another groun has reported that a transmembrane potential is i m p o f lant for translocation [8(l]. In Ref. !69 the reduction of the A-B disulfide l~md was examined. Using a thioredoxin system it was shown that the A-B disulfide is much more easily reduced in the low pit conformation than in the nati.'e conformation. This indicates i'hat this disulfide is relatively buried uutil exposure of the toxin to low pH. However. this

45 Ixmd was cleaved by rcductants such as D l ' r or glutathione in the native state at net,,r:l! pH. ~.rcsumabl~, because their small size allows access to the disulfide. Cellular reduction of the A-B disulfide on plasma membrane bound toxin was also demonstrated. The rcductk, n only occurred afte, expo.,,nrc to low external pH. This suggested that toxin penetrated the plasma membrane in an orientation such that the A-B disulfide became exposed to the cytoplasm However. only 5-10% of all toxin molecules became reduced. This may mean that most of the bound toxin molecules arc in some Ixml that does not lead to translocation. For examplc, there might bc rome hctcrogcncity in the receptor Impuhltion that wc do not yet appreciate. Another possibility is that there was inefficient translo cation due to heterogeneity in the nicking site (,,t'e bclow). In the next study direct biochemical evidence was tfl~taincd to demonstrate that after low pH treatment of cells with toxin Ixmnd to thc plasma membrane the A fragment reached the cytoplasm [182]. It was fimnd that after low pH triggered insertion into the plasma membrane two toxin fr gmcnts wcrc protected from digestion hy externally added pronase E. ProK-tion involved 5- I(IC4 of the [stand toxin, suggesting that the same mcmbranc-inscrted population susceptible to reduction was involved. One protected fragment, of 2(I kDa, was idcntificd as the A fragment by its ability to ADP-ribosylatc EF-2. The other protected fragment. !S kDa. was derived irom the B domain. When sal~min was used to permcabilizc the Vcro cell membrane, only the A fragment leaked out. "]'his implied the A fragmcnt had been frcc in the cytoplasm prior to ~,aponin treatment. The B domain fragment rcmainml membrane bound and w~.,~oat 'clea.',ed. Its hydrophobicity was confirmed by Tr:.,',on X-114 binding using the phase partititm assay [172]. Interestingly, the degree of translocation was strongly temperature dcpendcn,, being much more efficient at higher tcmpcratures. Since cndocytosis is highly temperature dependent, it was suggested thpt mmc residual unblocked endocytosis occurring at higher temperatures could explain this result. Anoth~,r Ix~ssibility is that translocation is conformation-dependent, with the A domain unfiflding that occur.,, at higher temperatures necessary fi~r translocation [I 12] (scc section V). in Ref. 183 evidence that protcolylie proccssing of the C-terminal of the A fragment affects lranslocafion was prcsc:ded. Thrcc major variants of fragment A were found ha~ing similar molecular weights, but different charge. These wcrc identified with fragment A cleaved alter ART-190. 192 and 193. This interpretation was supported by the observation that carboxypcplidase B progressively converted the variants into the most acidic form (clem,cd after ART-190). Using a mixture of forms it was shown that after binding of toxin to

cells only the most acidic vartant was translocated into the cytosol. One pos,,ibility was that this resulted from proteolysis occt:rring after translocatmn into the cytosol. This ,'~ssibility was eliminated by an experiment showing car~xypeptidase treatment of toxin before addition to cells increased translocatitm. Cart~xypeptidasc prctrcatment also increased pore formation by the toxin. In an, .her study, it had been observed that labeling of fragment A by hydmphobic photolahcling agents was greatest for a smaller variant of the A fragment [151]. T,)gcthcr, these results suggest that there may actually b c a difference in the way C-terminal procesmd and unprocessed fragment A interacts with the membrane. It would bc interesting to know whether the processing of the C-terminal perturbs the equilibrium between the fl~lded/hydrophilic fi~rm and the unfiflded/hydrophobic fi~rm of fragment A [I 12] (see section V). The toxicity of hybrids made from two different ,,oxins cain also yield information about the translocation process [184,185]. in one rel'x~rt it was demonstraled ~hat a diphtheria toxin B fragmcnt-ricin A chain conjugate was toxic to cells [185]. Interestingly, this hybrid required low pH ft~r entry, as shown by its sen.qtivity to NH~CI inhibition, and by its toxicity when I~)und to the plasma membrane and exposed to low pH. Thi.~ sl,gge,~ts that the B domain has a major role in the translocation process at low pl I and that there is ao absolute specificity fi}r a particular A domain. Another observation was that the hybrid had the cell bmding properties of diphtheria toxin. This is consistent with the observation that the B domain [x~ssesscs the receptor binding site. Further evidence that there i.~ no absolute specificity fiw the A domain come., from studies of the transhx:ation of mutant toxins contaip;ng peptide extensions on their N-terminus of the A domain [18~,]. Pn)nasc digestions and saponin-dcpendent release indicated that the extended A domains could insert and tran:aocatc properly. O n e caution in studying translocation across ceil membranes is that the exact status of the endocytotic process is not always clcar. O n e mcth{~l used to stop entry via endosonlcs is to ado agents that raise cndo:-x~real pH. However, the functioning of thcsc inhibitors when external pH is low has not been fully characterized. It may also 0c a problem that the low temperalure somelime,~ used to stop ,.-nd~'ytosis may directly affect the toxin. Another limitation of plasma membrane translocation studies is that any specific endosoreal proteins that play a special role would be missed. Finally. this system is often limited to analysis by cytotoxicity or gei electrophorcsis. Nevertheless, the plasma membranc sysicni has been one of the most valuable and informative systems for the analysis of toxin entry into cells.

46 W i l l . Diphtheria toxin receptor:, identity and possible effects on toxin behavior

The identification of the cellular receptor for diphtheria toxin has proven to bc very difficult. The early specular;on that lipids could act as the toxin rcccptor has received little support (me section XI). in other early studic,; a glycoprotcin that bound toxin strongly was identified in CHO cells [187]. However, it has not been demonstrated that this protein is the functional diphtbei;a toxin receptor. Recen! work has concentrated on identifying the high affinity, functional receptor on Veto and BS-C-I (monkey kidney) cells [188]. Some studies raised the possibility that an anion transport protein was the functional receptor. It was dcmonstr ited that toxin inhibited anion transport [189], and that anion transport was necessary for toxin entry. [190], with inhibition of toxin ent.r:,' ,iccurlit;g upon addition of inhibitors of anion transialrt. However, there are several factors that complicate interpretation of the interaction of anion transport and toxin entry. The anion transporter appears to bc involved in regulation of cellular and vacuolar pH, and anion transport in cells may be indirectly affected by the cation transport induced by the toxin [191-194]. It was al~) found that the anion effect on toxin might only involve inhibition of translocation by an outwardly directed anion gradien; [195]. In addition, there has been no direct evidence to prove an anion transporter protein binds toxin. Thus, the role of anion t~ansportcrs in toxin entry remains incompletely undcrstcmd. Nevertheless, it remains possible that the anion transporter is a component of the toxin reccptor, perhaps in combination with the receptor protein de~ribed below The identification of a low molecular m;,ss (15-20 kDa) protein that acts as , receptor for diphtheria toxin was reported by two groups [196,1t~7]. This protein was identified as a functional receptor by several criteria. First, after binding toxin to t-'Sl-labeled cells,,. the 15-211 kDa protein coulzl be co-immunoprccipitatcd with the toxin. This protein could also be efficiently chemically crosslinked to L'51-1abeled toxin. Crosslinking could be competed away with an excess of unlabeled toxin. Furthermore, toxin binding to this protein could be competed away by toxin ligands such as IHP and ATP. These ligands can inhibit toxi, action on ~nsitive cells [41,461. In the con;cx;, of this rc~,icw the most important question about thc receptor of diphtheria toxin is the role that it play:; in the translocation process. Basically, the question to be answered is whether the receptor simply acts as a carrier that must bring the toxin to the correct endosomal compartmem or whether it dircttly influences the toxin during the translocation step. The fact that translocation can be observed in vitro in the absence of other proteins suggests that the receptor

docs not play an absolutely essential role. However, this may be somewhat deceptive, since we do not yet know whether translocation in vitro and in vivo arc identical. The question of receptor function has been examincd experimentally using surrogate molecules to provide cell surface binding. It has been shown that intoxication does not occur after low pH exposure of biotinylatcd toxin bound to cell surface associated avidin [198]. in addition, upon exposure to low pH the avidin bound toxin does not penetrate the plasma membrane the same way as toxin bound to its cellular receptor. Therefore, it has been argued that the receptor must provide a function in addition to binding. However, interpretation of the.it: experimcms is difficult without knowing whether avidin is ~,aund directly to the plasma mombrane or an extracellular protein, and whether the biotin-avidin link ix ,~c,ik enough to disst~:'iatc upon ex~)sure to low pH (pH 4.8). Another app,mach has been to compare the cytotox-. icity of wild type toxin to that of a mutant that lacks thc ability to bind the rcccptor and is conjugated to a cell surface binding a,.tibody [199]. In this case the mutant-antibcn:ly conjugate showed full toxicity and similar cytotoxieity kinetics to wild type toxin. Therefore, it was argued that the receptor must only function ;o efficiently, carry the toxin to the endo~me. However, in a seet;nd study a mutation at Pro-308 was found to disrupt translocation of an anti~xly-conjugatetl toxin but not of unconjugated toxin [2(X)]. "l'hercfarc, the issue of the role of the receptor in translocation remains complex. This is likely to remain the case until the receptor is characterized and i~dateo so that the interactions between it and the toxin can be directly examined. XIX. Model for translocation oF diphiheria tox;'~ Now that studie.., of Ihc structure, membrane in.~rlion and translocafion of the toxin have been described it is wortilwhile t,~ c,m~!der how translocation might occur. [At us first consider the translocation mechanism for a single toxin molecule, starting at lhc point in which it encounters tile low pH of the endosome lumen. D,w pH would induce the partial unfolding of the toxin, so that it becomes hydrophobic and inserts into the ,nembranc. The phase diagram in Fig. 3 suge,c.,it.~ ;hat at 37~C and pl-1 :to,it 3 both the L' (A domain folded) and L" (A domain unfolded~ states may b2 present ~'e do not know whether low pH relearn,, toxin from its receptor ai ~is time. One medici for transh.,cation would involve molecules in the L" state, in the L" state the toxin might take on something lesembling the cleft/partial wrapper structure and perhaps form a small pore. Alternately, pore formation could be due to any toxin p r e ~ n t in the L'

47

ENOOSO/~AL tUMEN pH~$.3

~.

~

XX. Comparison of Pseudomonas exotoxin A behavior to that of diphtheria toxin

CYTOPtASM oh~7 5

A. N

k"

~vDe~uC

I

~tM~!

cvlO~A$~lvOwAm~ C~AC!

8

O,$UlFIO~ e(rotc~,~,c,

1

/

/

Fig. 6. Schematic illuslration of the steps in a ccgfilrmahon-spccific translocation process. Set' text fl)r details.

state, as this conformation contains the hydrophilic folded A domain which might not be aHe to fit into the "hole' formed by a memt rane-in~rted B domain. There could a l ~ be a transient permeability increase due to membrane oisruption during membrane in~rtion. The exposure of membrane-'.'nserted toxin in the L" state to the neutral pH cytoplasm should cause the B domain to become less hydrophobic and the A domain to become hydrophilie (R" state, ~ e Fig. 2 (bottom)). As a result the degree of contact with the membrane would decrease and the A domain c,~uld move toward the ~,toplasm. Perhaps the pore, or a larger pore forms as the A domain leaves the membrane at this time. In addition, the A-B disulfide could reach the cytoplasmic side and be reduced by glutathionc at this time. The A fragment would then be released from the membrane and would ref~,ld iato it:~ active form. This model is summarizcd in Fig. 6. An alternate model is that the A domain crosses membranes without contacting lipid through toxin-induced pores or membrane disruption, in an extreme case with a large enough pore, or ~vere eno~'.gh membrane disruption, leakage of glutathionc and breakdown of endosomal oH could first result in release of A Iragmcnt w~thin the endosomal lumen. Its translocation might then oc,:ai even without it ha,,nng to unfold. These processes are mo~t likely to occur at high toxin concentration where, perhaps due to oligomer formation, membrane disruption, pore n,nmber, and pore size are likely to be the greatest.

it is worthwhile to briefly compare the behavior of diphtheria toxin with that of P~e,Momo,as cxo~oxin A, another toxin which catalyzes ADP-rihosylati,~n of EF2. Several groups have fotmd that there is ~quence homology between the catalytic domains of the L~,o toxins [57-62], but thcrc is none between the rest of their sequences. The exotoxin is also different fr~m diphtheria toxin in its total lack of identifiable continuous hydropl',obic stretches [63l and in its binding to a different receptor [201]. X-ray crystallography has identified three domains in the cxotoxin [63]. The N-terminal domain is involved in receptor binding, the C-terminal domain is the catalytic domain, and by largely default the central domain has been associated with transloeation [202,203]. However, recent studies have indicated that all three domains become hydrophobic at low pH, suggc,,,fi,g that all may participate in translocation [204]. Interpretation of the domain structure of the cxotoxin is comphcated by a probable proteolytic processing site at Arg-279 which divides the protein into two almost equal pieces. Several observations suggest this site is important ano that it is analogous to the nicking site which divides diphtheria toxin into A and B domains. Most importantly, proteolysis at this site has been shown to be nccegsary for toxicity [205]. In addition, the ~qucnce around this site fits *.he consensus for a vacuoiar proteol.vsis site [1~3]. Also, it is bridged by a disulfide bond as in diphtheria toxin. Finally. it is at a point where the ~quence bias of the cxotoxin changes noticeably (scc section VI). On it:~ C-tcrrainal side the protein is unusually Lys and His ptmr relative to that on its N-terminal side [60,118]. II1 vitro low pH ind:~ces a conformatio,al change in the cxotoxin, renders it hydrophobic, and allows ,t Lo penetrate into model mcmbrancs [2(16-209]. However, it may be that low pH does not directly !rigger membrane in~rtion in vivo. It has been found that the exotoxin hndergoes two distinct conformational changes at low ptl. and the change that occurs in the rangc of endo.soma[ pH does not render it very hydrophobic [129]. Instead. it is possible that cndosomal pH renders the toxin ~nsitive to somc proccssin~ stcn, such as the protcolytic cleavage described above, it could be that proteolysis makes the exotoxin hydrophobic by acting in combination with low pH or ~ m e other event, in this regard it is interesting that the endosome ma~, not be the site of membrane translocation oi" the exotoxin [210]. On the other hand it is also possible that factors missing n~: vitro, such as specific lipids or receptor binding, alter the exotoxin to aih,~' it to membrane insert at endosomal pH without additional processing. Further compan,;on of the behavior of diphtheria toxin

48 and exotoxin A should reveal much about hov, It)w pH and other factors cur .ine to allow translocatiott.

XTI. ,~aralJels between diphthe."ia toxin memhrane translocation and the membrane traaslocation of ordinary cellular proteins One of the interesting questions we can ask about the toxin is what lessons its behavior may have for translocation of ordinary cellular proteins [14]. It is likely that the different n, echanisms by which a proteir can cross a membrane wi!l have much in common, so toxin translocation sh,mid tell us much about transloC~t_in~ in ~enerai. On the other h a n d : t h e toxin encodes much. if not all, of the information for translocati.m within a single polypeptide, and does not need a translocation process consistent with maintenance of normal cellular function. For this reason it may tak( ~ m e 'short cuts' not found in normal cellular translo.. cation. Nevertheless, models for translocation suggest striking parallels between toxin and normal cellular translocation. One of the first models for texin translocation envisioned a pore allowiag translocation of an unfolded fragment A. This closely resembles the model proposed for ordinary secreted proteins [211]. The observation that the A fragment may contact the bilayer, which suggests a role for direct interaction nf a translocating protein with lipid, parallels the ideas of the membrane trigger hypothesis for cellular protein translocati~n [212]. Furthermore, the proposal that partial unfolding of fragment A may be necessary for its membrane insertion and translocatior, is similar to the recent realization that ordinary proteins must remain in an incompletely folded state it, order to be translocated [213,214]. It is also fa~inating that in recent studies parallels have been found between the behavior of the toxin and SecA. the protein in E. coil that appears to function in engaging precursor proteins with the membrane [215]. It has been found that SecA switches between sol,tble and membrane-inserted forms, and that a partial unfolding event is probably involved in this change (Ulbrandt, N.D., London. E. and Oliver, D.B., unpublished observations). Clearly. lessons learned from toxin translocation promise to continue to provide insight into protein translocation in generai.

XXli. Remaining questions and future r~earch There are several obvious questions that remain concerning the structure and function of diphtheria toxin. First is the question of what is its three-dimen. sional structure, it is likely ;hat the solution ,structure o f the native toxin will be known .soon. However, this will not be enough. We also need to derive a detailed

J knowlcdg: of toxin structure in m e m b r a n e s Therefore, ana!y~is of the topography and quaternary structure of t?,e membranc-in.sertcd toxin will remain an important i.~c',s of research, h= this regard, site-directed mutagenesi,; "rod the increa~:ing arr;y of techniques available to explore the properties ot mutant proteins should be at= ;nvaluable aid [! J5,216]. ~n terms of the '~ther cellular factors that may influc:x e the entry palhway just as much must be done. A :'uller kncwlcdgt of the toxin receptor and its role in translocation is badly needed. In addition, we must fiad out whether any endosomal or other proteins have a direct interaction with the toxin that aids in its Iranslocation. In conclusion, it is worth repeating that diph:heria toxin and related toxins form a fascinating area of research both because of the implications tor understanding normal cellular processes and the potentially enormous biomedical applications of toxins. The combinaiion of cell biological and biochemical/biophysical approaches has. and should continue, to help decipher diphth~'-;" ~..':'xinstructure and function. Whea a single model fully accounts for the behavior of the toxin both in cells and in vitro we will understand its membrane interaction and translocation. Although we :annot yet claim ',o be close to the final answer, significant progress has already been made and an exciting period of toxin research is underway. Acknowledgement This work was supported by N I H grant GM 31986. Relerences I Pizza. M., Barloloni, A., Prul;noh*, A.. S:k'cslri, S. and Rappuoli. R. (1¢)88) Pr(x:. Nail. Acad. Sci. USA 8.