title: A study of the YopD-lcrH interaction from Yersinia pseudotuberculosis reveals a role for hydrophobic residues within the amphipathic domain of YopD.
abstract: The enteropathogen Yersinia pseudotuberculosis is a model system used to study the molecular mechanisms by which Gram-negative pathogens translocate effector proteins into target eukaryotic cells by a common type III secretion machine. Of the numerous proteins produced by Y. pseudotuberculosis that act in concert to establish an infection, YopD (Yersinia outer protein D) is a crucial component essential for yop regulation and Yop effector translocation. In this study, we describe the mechanisms by which YopD functions to control these processes. With the aid of the yeast two-hybrid system, we investigated the interaction between YopD and the cognate chaperone LcrH. We confirmed that non-secreted LcrH is necessary for YopD stabilization before secretion, presumably by forming a complex with YopD in the bacterial cytoplasm. At least in yeast, this complex depends upon the N-terminal domain and a C-terminal amphipathic alpha-helical domain of YopD. Introduction of amino acid substitutions within the hydrophobic side of the amphipathic alpha-helix abolished the YopD-LcrH interaction, indicating that hydrophobic, as opposed to electrostatic, forces of attraction are important for this process. Suppressor mutations isolated within LcrH could compensate for defects in the amphipathic domain of YopD to restore binding. Isolation of LcrH mutants unable to interact with wild-type YopD revealed no single domain responsible for YopD binding. The YopD and LcrH mutants generated in this study will be relevant tools for understanding YopD function during a Yersinia infection.
authors.affiliation: Department of Cell and Molecular Biology, Umeå University, S-901 87 Umeå, Sweden.
authors.forename: M S
authors.initials: MS
authors.lastname: Francis
authors.forename: M
authors.initials: M
authors.lastname: Aili
authors.forename: M L
authors.initials: ML
authors.lastname: Wiklund
authors.forename: H
authors.initials: H
authors.lastname: Wolf-Watz
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins; D001483:Base Sequence; D017931:DNA Primers; D018832:Molecular Chaperones / Q000378:metabolism*; D011485:Protein Binding; D020798:Two-Hybrid System Techniques; D015011:Yersinia pseudotuberculosis / Q000378:metabolism*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D017931:DNA Primers; D018832:Molecular Chaperones; C118339:SycD protein, bacteria; C078910:YopD protein, Yersinia