Essential lysine residues within transmembrane helix 1 of diphtheria toxin facilitate COPI binding...

Essential lysine residues within transmembrane helix 1of diphtheria toxin facilitate COPI binding and catalyticdomain entrymmi_7159 1010..1019

Carolina Trujillo,1,2 Julian Taylor-Parker,2

Robert Harrison2 and John R. Murphy2*1Department of Microbiology, Boston University Schoolof Medicine, Boston, MA 02118, USA.2Section of Molecular Medicine, Department ofMedicine, Boston University School of Medicine,Boston, MA 02118, USA.

Summary

The translocation of the diphtheria toxin catalyticdomain from the lumen of early endosomes into thecytosol of eukaryotic cells is an essential step in theintoxication process. We have previously shown thatthe in vitro translocation of the catalytic domain fromthe lumen of toxin pre-loaded endosomal vesiclesto the external medium requires the addition of cyto-solic proteins including coatomer protein complex I(COPI) to the reaction mixture. Further, we haveshown that transmembrane helix 1 plays an essential,but as yet undefined role in the entry process. Wehave used both site-directed mutagenesis and a COPIcomplex precipitation assay to demonstrate thatinteraction(s) between at least three lysine residues intransmembrane helix 1 are essential for both COPIcomplex binding and the delivery of the catalyticdomain into the target cell cytosol. Finally, a COPIbinding domain swap was used to demonstrate thatsubstitution of the lysine-rich transmembrane helix 1with the COPI binding portion of the p23 adaptor cyto-plasmic tail results in a mutant that displays full wild-type activity. Thus, irrespective of sequence, theability of transmembrane helix 1 to bind to COPIcomplex appears to be the essential feature for cata-lytic domain delivery to the cytosol.

Introduction

In recent years the study of the molecular mechanism bywhich the diphtheria toxin catalytic domain is translocated

across the endosomal membrane and released into theeukaryotic cell cytosol has been the focus of much atten-tion (Lemichez et al., 1997; Oh et al., 1999; Ren et al.,1999; Ratts et al., 2003; Ratts et al., 2005). In 2005, Rattset al. conducted an in silico analysis of diphtheria toxin,which described the T1 motif in helix 1 of the transmem-brane domain. This motif was found in anthrax lethalfactor and oedema factor, and botulinum neurotoxinsserotypes A, C and D; wherein its location is conserved inregions of these toxins that are believed to first emergethrough a trans-endosomal membrane pore on the cyto-solic side of the vesicle during the catalytic domain entryprocess. Ratts et al. (2005) also demonstrated by pulldown experiments that T1 motif sequences bound at leastb-COP of the COPI complex. These observations con-firmed and extended earlier work that demonstrated thata cytosolic translocation factor complex, which includedb-COP, was required for the in vitro translocation of thediphtheria toxin catalytic domain from the lumen of acidi-fied endosomal vesicles (Lemichez et al., 1997; Rattset al., 2003).

COPI is a heptameric complex (a-, b-, b′-, g-, e-, z- andd-subunits) that facilitates vesicular retrograde transportbetween Golgi compartments, the Golgi apparatus andthe ER, and in endosomal vesicle trafficking (Serafiniet al., 1991; Waters et al., 1991; Whitney et al., 1995).COPI complexes are recruited to the membrane surfaceen bloc by Arf-GTP (Donaldson et al., 1992; Palmer et al.,1993). Following the initial binding of COPI to the mem-brane, additional interactions through secondary bindingof the complex to dibasic signatures (KKXX, KXKXX)and/or aromatic amino acid sequences [i.e. FFXXBB(X)n]that are present in the cytoplasmic tails of cargo andp23/24 adaptor proteins further stabilize binding to themembrane surface (Cosson and Letourneur, 1994; Harterand Wieland, 1998; Eugster et al., 2004).

Since the emergence of either the T1 motif or trans-membrane helix 1 sequences on the cytoplasmic side ofendosomal vesicle membranes could potentially mimiceither cargo motifs and/or the KKXX and FFXX of adaptorproteins that are known to bind COPI complex proteins,we have used both site-directed and domainswap mutagenesis to further study the role played by

Accepted 31 March, 2010. *For correspondence. E-mail [email protected]; Tel. (+1) 617 638 6014; Fax (+1) 617 638 6020.

Molecular Microbiology (2010) 76(4), 1010–1019 � doi:10.1111/j.1365-2958.2010.07159.xFirst published online 26 April 2010

© 2010 Blackwell Publishing Ltd

transmembrane helix 1 in the catalytic domain entryprocess. We demonstrate that at least three lysine resi-dues within this region of the toxin are essential for bothcytotoxic activity in vivo and to mediate binding to COPIcomplex proteins in vitro. Further, we demonstrate thatreplacement of the native transmembrane helix 1 with the13-amino-acid COPI binding domain from the cytoplasmictail of p23 results in a domain swap mutant whose cyto-toxic potency is identical to that of the wild-type toxin.These results suggest that COPI complex binding to theN-terminal lysine-rich portion of the transmembranedomain is an essential feature for catalytic domain deliv-ery to the eukaryotic cell cytosol.

Finally, we discuss a dynamic model of toxin entry inwhich we propose that as the lysine-rich region of trans-membrane helix 1 emerges from the trans-vesicle mem-brane pore it functions as a cargo and/or adaptor proteinCOPI binding site mimetic. Because the N-terminalportion of the diphtheria toxin transmembrane domain,with its disulfide bond-linked catalytic domain, appears topass unencumbered through the endosomal vesiclemembrane pore, the protein–protein interaction(s)between these lysine residues and COPI complex

appears to facilitate catalytic domain translocation anddelivery to the cytosol much like pulling a string through astraw.

Results

Based upon our earlier observations describing the rolethat the T1 motif and transmembrane helix 1 are likely toplay in the catalytic domain entry process and the well-defined cargo motifs recognized by COPI complex pro-teins, we initiated a site-directed mutational analysis ofthe multiple lysine residues in this region of diphtheriatoxin. Figure 1A shows a partial amino acid sequence ofdiphtheria toxin fragment B from Serine195 to Aspar-agine235 showing transmembrane helices 1 and 2, the T1motif and the multiple lysine residues that we anticipatedwere most likely to mediate binding to COPI complexproteins and thereby facilitate catalytic domain entry.Because cloning of wild-type diphtheria toxin is prohibited,we introduced site-specific mutations into the structuralgene encoding the fusion protein toxin DAB389IL-2.DAB389IL-2 is composed of native diphtheria toxin catalyticand transmembrane domain sequences, amino acids

Fig. 1. A. Following trypsin ‘nicking’ after Arginine194, native diphtheria toxin may be separated into an N-terminal ~21 800 Da catalyticdomain and the C-terminal ~36 900 Da transmembrane and receptor binding domains. A partial N-terminal amino acid sequence of thetransmembrane domain from Serine195 to Asparagine235 that includes transmembrane helices 1 and 2 and the T1 motif is shown.B. Effect of wild-type DAB389IL-2 and variant site-directed mutants in transmembrane helix 1 on [14C]-leucine incorporation into HuT102 cells.DAB389IL-2 (�); DAB(K213A)389IL-2 (�); DAB(K215A)389IL-2 (�); DAB(K217A)389IL-2 (�); DAB(K222A)389IL-2 (�).C. DAB389IL-2 (�); DAB(K213A, K215A)389IL-2 (�); DAB(K215A, K217A)389IL-2 ( ).D. DAB389IL-2 (�); DAB(K213A, K215A, K217A, K222A)389IL-2 ( ). Data points (mean � range) represent triplicate analysis at theindicated concentration from one representative experiment (n = 3).

Diphtheria toxin cytosolic entry 1011

© 2010 Blackwell Publishing Ltd, Molecular Microbiology, 76, 1010–1019

1–389, to which human interleukin 2 is genetically fused inthe correct translational reading frame (Williams et al.,1990). DAB389IL-2 has been shown to be selectively toxictowards eukaryotic cells that display the high-affinity IL-2receptor, and except for receptor binding specificity thefusion protein toxin has been shown to follow the sameroute of entry into the cell as native diphtheria toxin(Bacha et al., 1988; Waters et al., 1992).

We first introduced single K → A mutations at positions213, 215, 217 and 222, after which the mutant fusionprotein toxins were expressed in recombinant Escherichiacoli and partially purified as previously described (vander-Spek et al., 1993). Individual mutant fusion proteins werethen examined for cytotoxic potency by dose–responseanalysis using high-affinity IL-2 receptor bearing HuT102cells as previously described (vanderSpek et al., 1993).As shown in Fig. 1B, the introduction of alanine site-directed substitution mutations at K213, K217 and K222results in a loss of 1–2 logs of cytotoxic potency relative tothe wild-type fusion protein toxin. In marked contrast, thesubstitution of alanine for K215 results in a mutant thatretains full cytotoxic activity. Lysine215 is positioned in themiddle of the palindrome KTKTK, and the mutantDAB(K215A)389IL-2 still retains three lysine residueswithin transmembrane helix 1.

These observations led us to predict that within trans-membrane domain helix 1 a combination of at least threelysine residues is most likely to be required for binding toCOPI complex proteins and subsequent delivery of thecatalytic domain. Furthermore, the spacing betweenthese residues appears to affect the efficiency of catalyticdomain entry into the cytosol as reflected by the 2 logrange in cytotoxic potency of this group of single K → Amutants. If this were the case, then either the introductionof any pair of K → A mutations or the quadruple K → Amutation in transmembrane helix 1 should lead to a com-plete loss of cytotoxic activity. In order to test this hypoth-esis, we constructed the double mutants DAB(K213A,K215A)389IL-2 and DAB(K215A, K217A)389IL-2, and thequadruple mutant DAB(K213A, K215A, K217A,K222A)389IL-2. Following expression and purification,each mutant fusion protein was then assayed for cytotoxicactivity. As anticipated, both of the double K → A mutantsas well as the quadruple K → A mutant were found to benon-toxic (IC50 > 5 ¥ 10-7 M) (Fig. 1C and D).

Because we have previously demonstrated by pulldown experiments that transmembrane helix 1 and the T1motif mediate binding to at least b-COP of the COPIcomplex, we next asked whether it was either the aminoacid sequence of transmembrane helix 1 and the T1 motifor simply the ability to bind to COPI complex that was theessential feature to mediate the cytosolic delivery of thecatalytic domain. Because the COPI binding motifs ofGolgi and ER cargo and adaptor proteins have been well

characterized, we constructed a domain swap mutant inwhich the 13-amino-acid COPI binding segment from thecytoplasmic tail of the p23 adaptor protein was used toreplace that portion of transmembrane helix 1 that con-tains the multiple lysine residues essential for catalyticdomain delivery to the cytosol.

The structural gene encoding DAB389IL-2 was modifiedsuch that amino acids 212–223 that encompass trans-membrane helix 1 were deleted and replaced with the13-amino-acid sequence encoding the COPI bindingportion of the p23 cytoplasmic tail (REILKKAKFFRRL).Following its genetic construction, the plasmid encodingthe mutant toxin DAB(212p23)389IL-2 was cloned,sequenced to verify correct insertion and reading frame ofthe COPI binding segment, and the recombinant mutantprotein was expressed and purified as described inExperimental procedures. As shown in Fig. 2, dose–response analysis of DAB(212p23)389IL-2 on Hut102 cellsis identical to that of the wild-type DAB389IL-2(IC50 ~ 10 pM). The functional equivalence between thelysine-rich transmembrane helix 1 and the COPI bindingsegment from the cytoplasmic tail of the p23 adaptorprotein suggests maintenance of the COPI complexbinding function of this region is an essential feature in thecatalytic domain entry process.

Based upon earlier reports from this laboratory as wellas studies examining the role of cargo motifs in COPIcomplex interactions, we next employed both GST pulldown experiments and precipitation assays to examinethe ability of wild-type and mutant transmembrane helix 1sequences to interact with COPI complex proteins. It hasbeen previously demonstrated both in vitro and in vivothat dilysine, poly-arginine and diphenylalanine motifs inthe cytoplasmic regions of Golgi and ER cargo proteins

Fig. 2. Effect of DAB389IL-2 and the COPI binding domain swapmutant DAB(212p23)389IL-2 on [14C]-leucine incorporation intoHuT102 cells. DAB389IL-2 (�); DAB(212p23)389IL-2 (�). Data points(mean � range) represent triplicate analysis at the indicatedconcentration from one representative experiment (n = 3).

1012 C. Trujillo, J. Taylor-Parker, R. Harrison and J. R. Murphy �


play an essential role in recognition and binding by COPIcomplex proteins. The proximity of numerous basic aminoacids within transmembrane helix 1 of diphtheria toxinprompted us to examine the potential role played by thesesequences in mediating the binding of COPI.

We first conducted a series of pull down experimentsin order to determine which subunit of the COPI complexwas likely to facilitate the binding between transmem-brane helix 1 sequences and the complex. The structuralgenes for g1-COP, b′-COP and e-COP were expressedindividually in vitro using a coupled transcription/translation rabbit reticulocyte lysate system. As shown inFig. 3, after in vitro synthesis and co-incubation witheither GST or GST-DT140–271, only GST-DT140–271specifically captured [35S]-labelled g1-COP. In contrastand in a manner consistent with other reports describingCOPI binding using either cargo or adaptor proteins, wefailed to selectively pull down detectable amounts ofeither [35S]-e-COP or [35S]-b′-COP using GST-DT140–271 as bait. These results suggest that both g1-COP inaddition to the previously identified b-COP (Ratts et al.,2005), but neither b′- nor e-COP are likely to mediatethe protein–protein interaction(s) with transmembranehelix 1.

Because the results from both mutational analysis andGST-DT140–271 pull down experiments suggested that

residues in transmembrane helix 1 interacted with COPIin a fashion that was similar to the interactions betweenthe complex and both cargo and p23 adaptor proteins, wenext analysed the ability of individual peptides from thisregion to bind to COPI complex proteins (Table 1). Inorder to conduct these experiments, we used an assayoriginally described by Hudson and Draper (1997) andsubsequently used by Reinhard et al. (1999) to demon-strate that paired diamines in neomycin, as well as thedibasic motifs contained within the cytoplasmic tails ofp23/24 adaptor proteins [FFXXBB(X)n] are capable ofinteracting with at least two specific sites on COPIcomplex, and depending upon the spacing induce aggre-gation and precipitation of COPI complex in vitro. Thus, todemonstrate that lysine residues in transmembrane helix1 functioned like p23 adaptor and cargo motifs in theirinteraction with COPI, we utilized this precipitation assayto examine the ability of diphtheria toxin transmembranedomain helix 1-based peptides to bind to and precipitateCOPI complex proteins. Figure 4A shows that DTB5, asynthetic peptide corresponding to amino acid residues214–230 of the transmembrane domain, is able to induceaggregation and precipitation of COPI coatomer in vitro.In a similar fashion, the addition of increasing concentra-tions of the COPI binding peptide derived from the cyto-plasmic tail of the p23 adaptor protein to the reactionmixture also resulted in the dose-dependent precipitationof the complex in vitro (Fig. 4C).

In order to demonstrate that the e-amino moieties of thelysine residues in DTB5 mediated the binding to COPIcomplex proteins, 1,3-cyclohexanebis(methylamine)(CBM) was added to the reaction mixture in increasingconcentrations. As shown in Fig. 4B, the addition of thediamine containing CBM to the reaction mixture com-pletely blocked the DTB5-dependent precipitation ofCOPI complexes in vitro.

To further show that at least two pairs of lysine residueswere necessary to induce COPI aggregation and precipi-tation, we next tested a series of DTB5-related peptides inwhich pairs of lysine residues on either the N- orC-terminal ends were changed to alanine (KNOFF andKCOFF) or a peptide (KOFF) in which all five lysine resi-dues were changed to alanine in the precipitation assay.As anticipated, removal of either all lysine residues or

Fig. 3. Autoradiographic analysis of GST and GST-DT140–217 topull down reaction mixtures following in vitro protein synthesis ofg1-COP, b′-COP and e-COP in rabbit reticulocyte lysates in thepresence of [35S]-methionine. Pull down reaction mixtures weredissolved in SDS-polyacrylamide gel loading buffer,electrophoresed on SDS-polyacrylamide gels, and followingelectrophoresis, gels were autoradiographed. Data are presentedas the relative binding of [35S]-labelled protein to each probe asmeasured by pixel density using Scion software. The datapresented is plotted as mean � range of three independentexperiments for each COPI subunit respectively.

Table 1. Synthetic peptides used in this study.

Peptide Amino acid sequence MW Reference

DTB5 TKTKIESLKEHGPIKNKM 2081 This studyKNOFF TATAIESLKEHGPIKNKM 1967 This studyKCOFF TKTKIESLKEHGPIANAM 1967 This studyKOFF TATAIESLAEHGPIANAM 1796 This studywtp23 EILKKAKFFRRLHGPIKNKM 2454 This study



lysine residues from either end of the peptide resulted inthe complete loss of COPI complex aggregation and pre-cipitation in vitro (Fig. 5).

Discussion

Lemichez et al. (1997) were the first to describe an in vitroassay to investigate the requirements for translocation ofthe diphtheria toxin catalytic domain from the lumen ofacidified endosomal vesicles to the external medium. Inthis assay system, endosomal vesicles were pre-loadedwith native diphtheria toxin in the presence of BafilomycinA1, and the early endosomal vesicle enriched fractionwas isolated by sucrose density gradient ultra-centrifugation. Upon removal of Bafilomycin A1, the addi-tion ATP to the reaction mixture allowed acidification of theendosomal lumen; however, acidification of the lumenwas not sufficient by itself to affect catalytic domain deliv-ery to the external medium. The addition of both ATP andcrude eukaryotic cytosolic extracts to the assay mixturewas found to be essential for catalytic domain transloca-

tion across the vesicle membrane and its release into theexternal medium. In addition, Lemichez et al. (1997)found that the addition of anti-b-COP to the in vitro assaymix was found to block catalytic domain translocationfrom the lumen of endosomal vesicles in vitro. Morerecently, using a bioinformatic approach, Ratts et al.(2005) described the presence of a conserved motif, T1(TKIESLKEHG), in the transmembrane helix 1 of diphthe-ria toxin. Because the cytosolic expression of a peptidethat carried the T1 motif in HuT102 cells was found toconfer resistance to the toxin and knock-down of peptideexpression restored toxin sensitivity, it was apparent thateither the T1 motif or transmembrane helix 1 of diphtheriatoxin is likely to play an essential role in the catalyticdomain entry process. In addition, pull down experimentsusing GST-DT140–271 demonstrated that at least theb-COP subunit of the COPI complex specifically bound toat least a portion of the T1 motif, and that this associationwas also essential for catalytic domain entry process.

In the case of diphtheria toxin, the T1 motif includes twolysine residues that may play a role in COPI binding and

Fig. 4. The aggregation and precipitation of COPI complex following exposure to the synthetic peptide DTB5 that carries the wild-typesequence of transmembrane helix 1. Partially purified COPI enriched fractions from bovine liver (2.5 mg of total protein/reaction) wereincubated for 1 h at room temperature with increasing concentrations of DTB5 (Table 1) in 40 ml of reaction buffer. The reaction mixture wasthen centrifuged (14 000 g, 30 min), and the supernatant fluid and pellet fractions were analysed by SDS-polyacrylamide gel electrophoresisand immunoblot using both anti-g1-COP and anti-b-COP as probes.A. Representative immunoblot analysis of COPI aggregation and precipitation following exposure to DTB5 (n = 4).B. Aggregation and precipitation of COPI complex proteins by 2 mM DTB5 in the absence and presence of increasing concentrations of1,3-cyclohexanebis(methylamine) (CBM) to the reaction mixture (n = 3).C. Aggregation and precipitation of COPI complex following exposure to the synthetic peptide wtp23 that carries the COPI complex bindingsequence from the cytoplasmic tail of p23 adaptor protein (n = 3). Immunoblot analysis was performed with only anti-b-COP antibodies.



catalytic domain entry; however, in anthrax lethal factorand anthrax oedema factor the separation of their respec-tive T1-like motifs from the multiple upstream KXKXXCOPI binding sequences raises addition questions as tothe role that the T1 motif per se may play in the entryprocess. To address this question we have begun a site-directed mutational analysis of the multiple lysine resi-dues in the N-terminal end of lethal factor in order todetermine the minimal sequence necessary for both COPIcomplex binding and delivery of LFnDTA to the eukaryoticcell cytosol.

In the present report, we have confirmed and extendedthese earlier observations and demonstrated that COPIbinding to the transmembrane helix 1 is dependent uponthe presence and spacing of at least three of the fourlysine residues, two of which are positioned immediatelyupstream of the consensus T1 motif (KTKTKIESLKEHG).We first conducted a site-directed mutational analysis oftransmembrane helix 1 in order to determine the minimalnumber of lysine residues that were necessary to facilitatecatalytic domain delivery to the eukaryotic cell cytosol.Following site-directed mutagenesis and DNA sequenceanalysis to ensure the introduction of each mutation, indi-vidual mutant recombinant proteins were expressed, puri-fied and tested for cytotoxic activity by dose–responseanalysis on HuT102 cells. With exception of a singlemutation (K215A), all of the single K → A mutant forms ofDAB389IL-2 displayed only a modest 1–2 log reduction intheir respective cytotoxic potency. In marked contrast, theintroduction of double K → A mutations (e.g. K213A,K215A or K215A, K217A) resulted in over a 5 log reduc-

tion in cytotoxic potency in their respective mutant fusionprotein toxins. Taken together the results of this site-directed mutational analysis has led us to conclude that aminimum of three lysine residues separated by three andfour amino acids were required for full cytotoxic potency;whereas, mutants that carried three lysine residues intransmembrane helix 1 that were separated by one andup to six amino acids retained cytotoxic activity; albeit withsome having reduced levels of activity. In the firstinstance, the e-amino groups of these lysine residuescluster on the same face of helical wheel projections;whereas, in the mutants with reduced activity the helicalwheel projections of the e-amino moieties show a greaterdistance along the vertical axis and distribution across theface (see Fig. S1). This observation suggests a degree ofdiversity of binding to COPI complex that is required fordiphtheria toxin catalytic domain entry to the cytosol issomewhat greater than has been previously described forcargo and/or adaptor protein binding and trafficking (Zer-angue et al., 2001; Eugster et al., 2004).

Hudson and Draper (1997) have previously demon-strated that at least two pairs of closely positioned aminomoieties in neomycin were necessary to cross-link andinduce precipitation of the COPI complex. In the presentreport, we have used a family of synthetic peptides whosesequences were related to transmembrane helix 1 andhave shown that the two KXKXX sequences carried bydiphtheria toxin transmembrane helices 1 and 2 are alsorequired to induce aggregation and precipitation of COPIcomplex in vitro. Because the addition of CBM to thereaction mixture completely blocked COPI aggregation

Fig. 5. Aggregation and precipitation of COPI complex following exposure to synthetic peptides that carry either wild-type transmembranehelix 1 and helix 2 sequences (DTB5), variants that lack either the N-(KNOFF) or C-terminal (KCOFF) dilysine signatures, or the syntheticpeptide in which all lysine residues have been replaced with alanine (KOFF). The assay was performed as described in Fig. 4, except thatfollowing the electrophoresis of supernatant fluid and pellet fractions, the immunoblots were only probed with anti-b-COP antibodies.



and precipitation in vitro, it is most likely that the interac-tion(s) between these toxin-related peptides and COPIcomplex is(are) mediated through free e-amino moietiesof the lysine residues within this region of the transmem-brane domain. Indeed, a similar requirement was seen byBéthune et al. (2006a) who defined the sequences fromthe cytoplasmic tail of the p23 adaptor protein that wererequired for COPI complex aggregation and precipitation.We recognize that COPI aggregation and precipitation inthe presence of these synthetic peptides does not neces-sarily reflect the conditions optimal for catalytic domainentry. However, taken together these experimentssuggest that the association between COPI and trans-membrane helix 1 is likely to be mediated through thee-amino moieties of lysine residues within this region ofthe transmembrane domain.

In order to further examine the role of transmembranehelix 1 and/or the T1 motif in the catalytic domain entryprocess, we reasoned that the functional ability to bindCOPI complex per se rather than the native amino acidsequence of this portion of the toxin transmembranedomain was likely to be the critical factor for the translo-cation and delivery of the catalytic domain to the cytosol.To test this hypothesis, we replaced transmembrane helix1 with the 13-amino-acid COPI binding sequence from thecytoplasmic tail region of the p23 adaptor protein. Theresulting COPI binding domain swap mutant,DAB(212p23)389IL-2, was found to be as toxic as the wild-type fusion protein toxin. It should be noted that the regionof transmembrane helix 1 that was exchanged with p23adaptor protein sequences contains the lysine residuesthat were shown to be essential for cytotoxic activity. Theresults presented here strongly suggest that at least onefunctional role of transmembrane helix 1 in diphtheriatoxin is that of COPI complex binding, and that thisprotein–protein interaction is essential for the delivery ofthe catalytic domain to the eukaryotic cell cytosol.

While we have not rigorously established which indi-vidual component(s) of the COPI complex mediates theinteraction with the lysine-rich region of the diphtheria toxintransmembrane domain, the pull down of [35S]-labelledg1-COP by GST-DT140–271 following in vitro transcriptionand translation suggests that this subunit along with b-COP(Ratts et al., 2005) is likely to participate in this interaction.The interaction(s) between transmembrane helix 1 of diph-theria toxin and COPI components appears to be unlike theinteractions mediated by the canonical dilysine signatureKKXX with a-COP and b′-COP (Letourneur et al., 1994;Eugster et al., 2004). However, cargo and adaptor proteininteractions with g-COP have been reported to exhibit morediversity in their binding profile including KKXX and KXKXXmotifs (Zerangue et al., 2001; Eugster et al., 2004), andresults presented here suggest this diversity of bindingmay be extended to transmembrane helices 1 and 2 of

diphtheria toxin as well. Despite the relatively well-characterized mechanism of COPI vesicle formation andfunction in retrograde traffic (for reviews see: Nickel andWieland, 2001; Béthune et al., 2006b), the role of COPIcomplex proteins in the trafficking of endosomal vesiclesremains largely unknown.

The molecular process by which the catalytic domainfrom DAB389IL-2 is delivered to the eukaryotic cell cytosolappears to follow the following steps: (i) binding of thetoxin to its respective cell surface receptor (Naglich et al.,1992), (ii) the internalization of the toxin::receptorcomplex by receptor mediated endocytosis into an earlyendosomal compartment (Moya et al., 1985) and (iii) uponacidification of the vesicle lumen by the (v)ATPase, thetransmembrane domain spontaneously inserts into theendosomal vesicle membrane forming an 18–22 Å poreor channel (Donovan et al., 1981; Kagan et al., 1981).Once the a-carbon backbone of the toxin is ‘nicked’ by theendoproteinase furin (Tsuneoka et al., 1993), the disulfidebond-linked C-terminal of the catalytic domain and theN-terminal end of the transmembrane domain appear tobe threaded in a fully denatured form into the pore formedby the membrane embedded transmembrane domain. Astransmembrane helix 1 and the T1 motif emerge on thecytosolic surface of the vesicle membrane, COPI complexis likely to recognize these sequences as p23 adaptorand/or cargo-like cytoplasmic tail mimetics and bind to thisportion of the transmembrane domain. However, unlikeeither the p23/p24 adaptor or cargo proteins that areanchored in the vesicle membrane, transmembranehelices 1–4 of the toxin with its disulfide bond-linked cata-lytic domain appear to be un-tethered and readily ‘pulled’through the transmembrane vesicle pore formed byhelices 5–9. Once the COPI complex facilitated delivery ofthe catalytic domain into the cytosol has occurred, wehave shown previously that thioredoxin reductase mostlikely reduces the disulfide bond between the N-terminalend of the transmembrane domain and the C-terminal endof the catalytic domain and at least Hsp90 appears tomediate refolding of the catalytic domain to a catalyticallyactive ADP-ribosyltransferase (Ratts et al., 2003).

Experimental procedures

GST and GST-DT140–271 pull down experiments

GST and the fusion protein GST-DT140–271 expression andpurification have been previously described by Ratts et al.(2003). Pull down experiments and HuT102 post-nuclearsupernatants were performed and prepared essentially asdescribed by Tamayo et al. (2008).

Synthetic peptides

Peptides used in this study are listed in Table 1. All peptideswere purchased from Kopella, Sudbury, MA and are amidated



on their C-terminus. The purity of all peptides as determinedby high-performance liquid chromatography was greater than90%.

Bovine liver COPI purification

COPI enriched fractions were prepared from bovine livercytosol as described by Waters et al. (1991) with some modi-fications described by Tamayo et al. (2008). The 13S fractioncontaining intact COPI was further purified by DE52(Whatman) column following manufacture’s specifications ina Biologic LP (Bio-Rad). Briefly, the DE52 cellulose wasequilibrated with 25 mM Tris-HCl (pH 7.4)/100 mM KCl, 1 mMDTT, 10% Glycerol. The column was eluted with a step gra-dient of 150, 500, 750 and 1000 mM KCl in 25 mM Tris-HCl(pH 7.4)/10% Glycerol/1 mM DTT. The elution correspondingto 500 mM KCl containing intact COPI complex and associ-ated material was dialysed against 25 mM Tris-HCl (pH 7.4),10% Glycerol and used as input material for the precipitationassays. During all steps the different fractions were analysedby immunoblot to confirm the presence of COPI components.Polyclonal antibodies to b-COP, g-COP, e-COP and z-COPwere from Abcam and S.C. Biotechnologies.

In vitro synthesis of COPI subunits

Vectors pCMV6-XL5 carrying the structural human genesencoding b′ COP (NM_004766.1), g1-COP (NM_016128.3)and e-COP (NM_007263.3) were purchased from OriGeneTechnologies (Rockville, MD). Full-length COPI subunitswere synthesized in vitro by using the TNT Quick CoupledTranscription/Translation System (Promega) in the presenceof [35S]-methionine following the manufacturer’s instructions(2 mg of plasmid DNA/100 ml of reaction volume). After in vitrosynthesis, the reaction mixture was diluted to 300 ml withbinding buffer [50 mM Tris-HCl, pH 7.4/150 mM NaCl/1 mMEDTA/1% Nonidet P-40/1¥ protease inhibitor cocktail(Roche)]. Pull down experiments with GST and GST-DT140–271 were performed as described above. Elutions were thenanalysed by SDS-PAGE and autoradiographed according tostandard methods.

COPI precipitation assays

HuT102 cells post-nuclear supernatant (25 mg of totalprotein) or partially purified COPI complex was incubated

with increasing concentrations of CBM (Aldrich 180467) orthe indicated concentrations of synthetic peptides in 40 ml ofreaction buffer containing 25 mM HEPES KOH pH 7.4,50 mM KCl, 25 mM Mg(OAc)2 and 1¥ protease inhibitor cock-tail (Roche) for 2 h on ice. The mixture was centrifuged(13 000 g for 25 min), and the supernatant fluid and pelletfractions were probed for b-COP or g1-COP by immunoblot.

Bacterial strains, plasmids and fusion toxin products

The parental plasmid pET-JV127 (vanderSpek et al., 1993)encoding for the fusion toxin DAB389IL-2 (AAA72359) wasused for cloning and purification of the mutant toxins. Theintroduction of the alanine exchange mutations and the p23adaptor COPI binding sequence swap was performed bysite-directed mutagenesis between the NsiI and RsiII restric-tion sites (Table 2). Briefly, synthetic oligonucleotides contain-ing the mutations were annealed and ligated into thepreviously digested parental vector. All mutations were con-firmed by sequence analysis using the sequence primer5′-GTCTCACTGAACCGTTGA-3′. All plasmids were propa-gated in chemically competent TOP 10 cells (Invitrogen), andprotein induction was done in the HMS174 (DE3) (Novagen)strain as described by vanderSpek et al. (1993). The syn-thetic oligonucleotides sequences used in this study areavailable upon request.

Purification of toxin mutant proteins

The induction of expression, inclusion body preparation andprotein purification was performed using a protocol previouslyreported by vanderSpek et al. (1993). Proteins were analy-sed by SDS-PAGE electrophoresis, 7% native gel electro-phoresis and immunoblot (Mo Anti DTA, Abcam). Proteinconcentrations were determined by the modified Bradfordreagent (Pierce). The percentage of full-length toxin in eachpreparation was determined by calculating the area under thecurve corresponding to toxin of silver stained 7% SDS-PAGEusing Scion Image for Windows software.

Cytotoxicity assay

Cytotoxicity assays were performed as described by vander-Spek et al. (1994). Figures were created in GraphPad Prismversion 5.01 for Windows, GraphPad Software, San Diego,CA, USA.

Table 2. Plasmids and IL-2 receptor-targeted fusion toxins used in this study.

Plasmid Tox gene product Amino acid sequence (212–225) Reference

pETJV127 DAB389IL-2 DKTKTKIESLKEHG vanderSpek et al. (1993)pETCT20 DAB(K213A)389IL-2 DATKTKIESLKEHG This studypETCT30 DAB(K215A)389IL-2 DKTATKIESLKEHG This studypETCT40 DAB(K217A)389IL-2 DKTKTAIESLKEHG This studypETCT50 DAB(K222A)389IL-2 DKTKTKIESLAEHG This studypETCT80 DAB(K213A, K215A)389IL-2 DATATKIESLKEHG This studypETCT90 DAB(K215A, K217A)389IL-2 DKTATAIESLKEHG This studypETCT60 DAB(K213, K215, K217, K222 → A)389IL-2 DATATAIESLAEHG This studypETCT70 DAB(212p23)389IL-2 EILKKAKFFRREHG This study



Acknowledgements

This work was supported by Public Health Service GrantAI021628 from the National Institute of Allergy and InfectiousDiseases (to J.R.M.) and by the National Institute of Allergyand Infectious Diseases New England Regional Center ofExcellence Grant AI057159.

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Essential lysine residues within transmembrane helix 1 of diphtheria toxin facilitate COPI binding...

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