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Nov 5, 1992 - (B) In vitro ADP-ribosylation of the rhoC protein. Lane 1, DC3AI-192B; lane 2, DC3A15192B; lane 3, DC3B; lane 4, DA1I7C3B; lane 5,. C3.
The EMBO Journal vol. 1 2 no.3 pp.921 - 931, 1 993

A chimeric toxin to study the role of the 21 kDa GTP binding protein rho in the control of actin microfilament assembly P.Aullo, M.Giry, S.Olsnes2, M.R.Popoff, C.Kocks1 and P.Boquet Unitd des Toxines Microbiennes URA CNRS 557, and 'Laboratoire de Gdndtique Moleculaire des Listeria, URA CNRS 1300, D6partement de Bacteriologie, Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris cedex 15, France, and 2Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway Communicated by S.Olsnes

We have developed a new tool for studying the role of rho in actin stress fibre formation. Clostridium botulinum exoenzyme C3 which affects actin microfilament assembly by ADP-ribosylation of p21 rho was genetically fused in various ways to diphtheria toxin (DT). The resulting chimeric toxins were tested on Vero cells. Chimeras of C3 and both the A and B fragments of diphtheria toxin had reduced cell binding activities but were apparently able to penetrate into Vero cells by the same mechanism as DT. Upon exposure to low pH, DC3B, a fusion protein of C3 and DT B fragment, had a high affinity for the DT receptor, but was apparently not able to translocate to the cytosol upon acidification. In spite of this, addition of picomolar concentrations of DC3B to the growth medium caused disruption of the cell microfilament system associated with vinculin and blocked cell growth efficiently, indicating that the C3 part of DC3B reached the cytosol, albeit by a different mechanism than that of whole diphtheria toxin. The chimeric DC3B toxin was also applied to Vero cells infected by Listeria monocytogenes, a pathogenic bacterium that uses an unknown mechanism of actin polymerization to move rapidly in the cytosol. DC3B inhibited the bacterially induced microfilament assembly indicating that L.monocytogenes utilizes a cellular rho dependent mechanism in this process. Key words: ADP-ribosylation/cytoskeleton/Listeria monocytogenes/membrane translocation

Introduction GTP binding proteins (G-proteins), which function by using guanine nucleotide binding and hydrolysing cycle (Bourne et al., 1990), are subdivided into three classes of molecules operating at distinct levels in cell regulation. The first recognized members of G-proteins were the ones involved in the control of protein translation: initiation and elongation factors (Weissbach and Ochoa, 1976). The second class consists of the heterodimeric G-proteins implicated in signal transduction (Stryer and Bourne, 1986). The last discovered class of proteins, binding GTP reversibly, is the ras superfamily of small GTP binding proteins (21 kDa) which includes more than 50 different proteins (Chardin et al., 1988). Small G-proteins, which are implicated in various a

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cellular regulations (cell growth, cytoskeleton assembly, intracellular traffic), have been classified into four different groups both by identity of structure and by their physiological activities: ras, rho, rab and arf (Hall, 1990). Rho proteins, first discovered in the marine mollusc Aplysia (Madaule and Axel, 1985), are subdivided into rho A, B and C (Chardin, 1988; Chardin et al., 1988; Yeramian et al., 1987) and Rho 1 and 2 in Saccharomyces cerevisiae (Madaule et al., 1987). The intracellular location of the mammalian rho proteins has been studied in both S. cerevisiae (McCaffrey et al., 1991) and in mammalian cells (Adamson et al., 1992). Proteins related to the rho proteins are present in both mammalian cells (racl, rac2; Didsbury et al., 1989) and in yeast (cdc42; Johnson and Pringle, 1990). In yeast, cdc42 is involved in the control of cell polarity (Johnson and Pringle, 1990), and rho in the control of the bud formation (J.S.Johnson, A.M.Myers, M.McCaffrey, P.Boquet and P.Madaule, submitted for publication). The racI protein is part of the enzymatic oxidase complex in neutrophiles (Abo et al., 1991). Racl has recently been shown to control actin ruffling in cells (Ridley et al., 1992). In higher mammalian cells, rho proteins have been implicated in the process of stress fibre assembly and disassembly (Chardin et al., 1989). This result has been obtained by using an ADP-ribosylating exoenzyme, purified from Clostridium botulinum C and D strains, called C3 (Aktories et al., 1987; Rubin et al., 1988), which selectively modifies the rho protein (Just et al., 1992). Upon cell microinjection of in vitro C3 ADP-ribosylated rhoA (Paterson et al., 1990) or application of ug/ml concentrations of the exoenzyme to cultured cells (Chardin et al., 1989), the first event subsequent to ADP-ribosylation of rho, was found to be a change of cell morphology with a selective loss of actin stress fibres detectable by fluorescent phalloidin derivatives (Chardin et al., 1989; Paterson et al., 1990). Conversely cell microinjection of mutated rho (Argl4-Vall4) which makes the protein permanently bound to GTP, induced stress fibre formation (Paterson et al., 1990). The use of microbial toxins, such as cholera, pertussis or diphtheria toxin, which ADP-ribosylate G-proteins, has indeed been of great help in understanding how these Gproteins operate in cells (Boquet and Gill, 1991). In order to use exoenzyme C3 as a reliable and efficient tool for the study of the physiological role of the rho protein, it was desirable to increase the efficiency of the translocation of C3 to the cytosol. We therefore decided to link by genetic fusion the C3 gene (Popoff et al., 1990) to DNA coding for diphtheria toxin which had been modified to have little or no intrinsic toxicity (Collier, 1990). A number of proteins have been shown to reach the cytosol in trace amounts, but the mechanism of translocation is incompletely understood. The molecules which enter the cytosol with the highest efficiency are certain toxins of 921

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bacterial and plant origin (Montecucco et al., 1991) among which diphtheria toxin (DT) has been most extensively studied in this respect (Sandvig and Olsnes, 1991). DT binds to a cell receptor located at the surface of sensitive cells (Naglich et al., 1992). After binding, DT is taken up by receptor mediated endocytosis. In the low pH environment of the endosome, a conformation change of the toxin B fragment (DTB) takes place, which allows the molecule to enter the lipid core of the membrane. The N-terminal 20 kDa enzymatic (ADP-ribosyltransferase) fragment A (DTA) enters the cytosol where it inhibits protein synthesis by ADPribosylation of the elongation factor 2 (EF2) (Collier, 1990). Recently, it was found that diphtheria toxin is able to translocate additional peptide and polypeptide materials into the cytosol with good efficiency (Stenmark et al., 1991; Wiedlocha et al., 1992). In the present study, we have made several constructs by fusing different parts of the DT gene with the C3 gene in order to obtain a maximally active chimeric toxin. The resulting molecules were then used to address the problem of how the rho protein can modify the assembly and disassembly of actin microfilaments.

Results Construction of chimeric toxins Membrane translocation of DTA could be facilitated by DTB in several ways. The different genetic constructions shown in Figure 1A, were made to test various possibilities. (i) The B fragment of DT by itself could contain enough information to allow the binding to the cell surface and translocation of DTA across the membrane as suggested by Kagan et al. (1981). DC3B corresponds to a single fusion between the C3 gene and the codon of the DT gene coding for the amino acid 175 which is located 17 residues before the end of the DTA fragment, thus retaining the first disulfide loop domain of DT which may be important for the translocation (Moskaug et al., 1987). (ii) A small peptide of about seven amino acids localized at the N-terminal end of DT fragment A could be required for the penetration of the enzymatic subunit of the toxin through the membrane (Chaudhary et al., 1991). DA-7C3B thus has the same general structure as DC3B except that an oligonucleotide coding for the first seven amino acids of DTA (Gly-Ala-Asp-Asp-ValVal-Asp) was added on to the 5' end of C3. (iii) DT fragment A could be acting cooperatively with the toxin B subunit in the process of membrane traversal as proposed in the 'cleft model' for DT translocation (Montecucco et al., 1991). To test this, DC3Aj - 192B was constructed by linking the C3 gene to the first codon of the full-length diphtheria toxin gene except for a point mutation in its enzymatic domain (Glu148-Serl48) which strongly reduces the toxicity (Collier, 1990). To explore if the first amino acids of DTA play a role in membrane translocation, DC3A15-92B was constructed in the same way as DC3A1- 92B except that in this case, another non-toxic mutant DT molecule (Glu148-Asp148) (Collier, 1990) was used which in addition lacked, by genetic deletion, the first 14 N-terminal amino acids. As shown in Figure 1B, all recombinant molecules had identical ADP-ribosyltransferase activities. By comparison with the C3 activity, we could not detect large differences between the chimeric molecules and the native C3 enzyme. Apparently, the fusion of other genes to either end of the 922

C3 gene, did not measurably impair the enzymatic activity of the corresponding protein. The different chimeric molecules were produced and purified as described in Materials and methods (Figure IC) and tested by Western blot using antibodies directed against either C3 or DT. As shown in Figure ID, the proteins obtained reacted with both types of antibody. The molecules were mainly 'un-nicked' in the sense that the disulfide loop between the A and the B fragments was not proteolytically cleaved since a major band of the molecular weight of the full-length construct was obtained in reducing gels (after trypsin treatment we obtained completely nicked chimeric proteins, data not shown). The breakdown products were not examined in detail. The ability of the chimeric molecules to bind to DT receptors was tested with Vero cells. Vero cells (from African green monkey kidney) are among the most sensitive cells to diphtheria toxin because they express a high number of DT receptors on their surface (Middlebrook et al., 1978). The binding of the chimeric molecules was assayed by measuring their competition with diphtheria toxin for DT receptors, thereby conferring protection against the toxic effects of DT, [measured as inhibition of protein synthesis (Uchida et al., 1973b)]. For comparison, we used a diphtheria toxin mutant, CRM197 (Uchida et al., 1973a) which is devoid of enzymatic activity but has retained full binding activity, especially in the nicked form (Uchida et al., 1973a). The results showed that the binding of the four chimeric molecules was quite different (Figure 1E). DC3B was found to have a high ability to compete with diphtheria toxin, even higher than nicked CRM 197, indicating that the Kd value of DC3B for the cell receptor was lower than that of nicked CRM197 (Mekada and Uchida, 1985). DA, 7C3B had a binding capacity of the same magnitude as nicked CRM197. DC3A1-192B and DC3A151-92B were poor competitors of DT for cell binding, which clearly indicates that these molecules recognize the DT cell receptor with lower affinity. From the results, we can estimate that the Kd of both DC3A1-192B and DC3A15-192B for DT receptor is 100-fold increased as compared with native DT (1.2 x O-9 M as reported by Mekada and Uchida, 1985). We also tested all the chimeric toxins after nicking by trypsin; no significant differences with the intact molecules in their affinities for DT receptor could be observed (data not shown). Altogether, the data indicate that the chimeric molecules have C3 enzymatic activity and bind specifically to the DT receptor albeit with different affinities. -

Activity of the chimeric toxins on cells To evaluate the activities of the four hybrid molecules in comparison with diphtheria toxin, we first tested their effects on cell growth. Cell growth is affected by inhibition of protein synthesis as well as by actin disassembly since in cytokinesis microfilaments are required to form the cleavage furrow (Schroeder, 1973). Indeed, it has been shown that upon treatment with large doses of C3, 3T3 cells were blocked in their cytokinesis (Rubin et al., 1988). Cells, seeded at low density, were treated either with the different chimeric molecules, with DT or with C3, and incubated at 37°C for 56 h. Thereafter, cell growth was measured by incubation with a dye measuring cell viability (Hansen et al., 1989). The test was performed both on toxin sensitive Vero cells and on resistant mouse L929 cells that lack specific DT membrane receptors. The effect on Vero

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