title: Intracellular plasma membrane guidance of Photorhabdus asymbiotica toxin is crucial for cell toxicity.
abstract: The bacterial toxin Photorhabdus asymbiotica toxin (PaTox) modifies Rho proteins by tyrosine GlcNAcylation and heterotrimeric Gα proteins by deamidation. Inactivation of Rho proteins results in F-actin disassembly in host cells. Here, we analyzed the subcellular distribution of PaTox and show that the glycosyltransferase domain of PaTox associates with the negatively charged inner surface of the plasma membrane. Localization studies with site-directed mutants, liposome precipitation analysis, lipid overlay assays, and confocal time-lapse microscopy revealed that a patch of positively charged lysine and arginine residues located on helix α1 of the glycosyltransferase is essential for membrane attachment. Using a helix1 deletion mutant, we show that plasma membrane localization of PaTox is essential for cytotoxicity and proved this by substitution of helix1 by an N-terminal myristoylation signal peptide, which restored plasma membrane localization and cytotoxicity. Furthermore, we also show that the intracellular deamidase activity of PaTox depends on the presence of the membrane localization domain. Comparison of PaTox membrane-binding domain with the 4-helix-bundle membrane-binding domain of Pasteurella multocida toxin, Vibrio cholerae multifunctional autoprocessing repeats-in-toxin, and clostridial glucosylating toxins revealed similar spatial geometry and charge distribution but different structural topology, indicating convergent evolution of toxin domains for optimized host target interaction.
authors.affiliation: *Departments of Experimental and Clinical Pharmacology and Toxicology and Pharmaceutical Technology and Biopharmacy, University of Freiburg, Freiburg, Germany; and Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany thomas.jank@pharmakol.uni-freiburg.de klaus.aktories@pharmakol.uni-freiburg.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Jank
authors.affiliation: *Departments of Experimental and Clinical Pharmacology and Toxicology and Pharmaceutical Technology and Biopharmacy, University of Freiburg, Freiburg, Germany; and Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany.
authors.forename: Christoph
authors.initials: C
authors.lastname: Trillhaase
authors.affiliation: *Departments of Experimental and Clinical Pharmacology and Toxicology and Pharmaceutical Technology and Biopharmacy, University of Freiburg, Freiburg, Germany; and Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany.
authors.forename: Natalia
authors.initials: N
authors.lastname: Brozda
authors.affiliation: *Departments of Experimental and Clinical Pharmacology and Toxicology and Pharmaceutical Technology and Biopharmacy, University of Freiburg, Freiburg, Germany; and Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany.
authors.forename: Marcus
authors.initials: M
authors.lastname: Steinemann
authors.affiliation: *Departments of Experimental and Clinical Pharmacology and Toxicology and Pharmaceutical Technology and Biopharmacy, University of Freiburg, Freiburg, Germany; and Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Schwan
authors.affiliation: *Departments of Experimental and Clinical Pharmacology and Toxicology and Pharmaceutical Technology and Biopharmacy, University of Freiburg, Freiburg, Germany; and Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany.
authors.forename: Regine
authors.initials: R
authors.lastname: Süss
authors.affiliation: *Departments of Experimental and Clinical Pharmacology and Toxicology and Pharmaceutical Technology and Biopharmacy, University of Freiburg, Freiburg, Germany; and Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany thomas.jank@pharmakol.uni-freiburg.de klaus.aktories@pharmakol.uni-freiburg.de.
authors.forename: Klaus
authors.initials: K
authors.lastname: Aktories
mesh_terms: D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D005798:Genes, Bacterial; D006367:HeLa Cells; D006801:Humans; D008563:Membrane Lipids / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010743:Phospholipids / Q000378:metabolism; D020637:Photorhabdus / Q000737:chemistry / Q000235:genetics / Q000472:pathogenicity*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity; D017384:Sequence Deletion; D055672:Static Electricity; D020741:rho GTP-Binding Proteins / Q000378:metabolism
chemical_list: D001427:Bacterial Toxins; D008563:Membrane Lipids; D010743:Phospholipids; D011994:Recombinant Proteins; D020741:rho GTP-Binding Proteins
keywords: Rho GTPase; deamidation; glycosyltransferase; heterotrimeric G protein; membrane localization domain