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

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

doi:10.1074/jbc.m406903200

Abstract

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.

Highlights

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 (LTcyt; 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
In the case of Rac, membrane association has been shown to favor the catalysis of GDP/GTP exchange by guanine nucleotide exchange factors and GTP hydrolysis by GTPaseactivating proteins [19, 26, 27]

Summary

EXPERIMENTAL PROCEDURES

Lipids and Reagents—Liver phosphatidylethanolamine (PE), brain phosphatidylserine (PS), liver phosphatidylinositol (PI), brain phosphatidylinositol 4,5-bisphosphate (PIP2), egg phosphatidylglycerol (PG), cholesterol, and synthetic lipids were from Avanti Polar Lipids. GDI Removal Protocol—The complex between GDI and Rac-GDP (1 ␮M) was incubated for 40 min at 30 °C with liposomes (6 mM lipids) and 20 ␮M GTP in buffer A supplemented with 1 mM MgCl2 and 2 mM EDTA to give a [Mg2ϩ]free of 1 ␮M. The pellet was washed with cold buffer A ([Mg2ϩ]free ϭ 1 ␮M), centrifuged again for 5 min at 4 °C, and resuspended in buffer A supplemented with 1 mM MgCl2 and no EDTA ([Mg2ϩ]free ϭ 1 mM). Liposome-bound Rac-GDP, which was obtained by the GDI removal protocol, was diluted 2-fold in buffer A supplemented with 1 mM MgCl2 and 1 mM MnCl2 to give a final lipid concentration of 3 mM and a final protein concentration of ϳ0.25 ␮M (as estimated by SDS-PAGE). The glass coverslips were mounted in Mowiol (Calbiochem and VWR International) and observed by fluorescence microscopy using a confocal laser-scanning microscope (Leica)

RESULTS

Lethal Toxin Binds to Phosphatidylserine

DISCUSSION