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J. Biol. Chem., Vol. 279, Issue 48, 49876-49882, November 26, 2004

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A Phosphatidylserine-binding Site in the Cytosolic Fragment of Clostridium sordellii Lethal Toxin Facilitates Glucosylation of Membrane-bound Rac and Is Required for Cytotoxicity^*

Bruno Mesmin, Karine Robbe¶, Blandine Geny||, Frédéric Luton**, Gérard Brandolin, Michel R. Popoff||, and Bruno Antonny¶¶

From the CNRS, Institut de Pharmacologie Moléculaire et Cellulaire, 660 Route des Lucioles, 06560 Valbonne, France, the ^||Institut Pasteur, Unité Bactéries Anaérobies et Toxines, 28 Rue du Dr Roux, 75724 Paris Cedex 15, France, and the Laboratoire de Biochimie et Biophysique des Systèmes Intégrés, Unité Mixte de Recherche 5092, Commissariat à l'Energie Atomique-CNRS, Université Joseph Fourier, 17 Avenue des Martyrs, F-38054 Grenoble Cedex 9, France

Large clostridial toxins glucosylate some small G proteins on a threonine residue, thereby preventing their interactions with effector molecules and regulators. We show that the glucosyltransferase domain of lethal toxin from Clostridium sordellii (LT_cyt; amino acids 1–546), which is released into the cytosol during cell infection, binds preferentially to liposomes containing phosphatidylserine as compared with other anionic lipids. The binding of LT_cyt to phosphatidylserine increases by two orders of magnitude the rate of glucosylation of liposome-bound geranyl-geranylated Rac-GDP. Limited proteolysis and deletion studies show that the binding site for phosphatidylserine lies within the first 18 N-terminal residues of LT_cyt. Deletion of these residues abolishes the effect of phosphatidylserine on the activity of LT_cyt on liposome-bound geranyl-geranylated Rac-GDP and prevents the morphological effects induced by LT_cyt microinjection into various cells, but it does not affect the intrinsic activity of LT_cyt on non-geranyl-geranylated Rac-GDP in solution. We conclude that the avidity of LT_cyt for phosphatidylserine facilitates its targeting to the cytosolic leaflet of cell membranes and, notably, the plasma membrane, where this anionic lipid is abundant and where several targets of lethal toxin reside.

Received for publication, June 21, 2004 , and in revised form, September 21, 2004.

^* This work was supported in part by grants from the Institut Pasteur (to M. R. P.) and the CNRS/Ministère de la Recherche (Actions Concertées Incitatives) (to B. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These authors contributed equally to this work.

^¶ Supported by la Ligue contre le Cancer.

^** Supported by INSERM.

To whom correspondence may be addressed. Tel.: 33-1-4568-8307; Fax: 33-1-4061-3123; E-mail: mpopoff{at}pasteur.fr. ^¶¶ Supported by the European Molecular Biology Organization Young Investigator Program and to whom correspondence may be addressed. Tel.: 33-4-9395-7772; Fax: 33-4-9395-7710; E-mail: antonny{at}ipmc.cnrs.fr.

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