title: Docking and assembly of the type II secretion complex of Vibrio cholerae.
abstract: Secretion of cholera toxin and other virulence factors from Vibrio cholerae is mediated by the type II secretion (T2S) apparatus, a multiprotein complex composed of both inner and outer membrane proteins. To better understand the mechanism by which the T2S complex coordinates translocation of its substrates, we are examining the protein-protein interactions of its components, encoded by the extracellular protein secretion (eps) genes. In this study, we took a cell biological approach, observing the dynamics of fluorescently tagged EpsC and EpsM proteins in vivo. We report that the level and context of fluorescent protein fusion expression can have a bold effect on subcellular location and that chromosomal, intraoperon expression conditions are optimal for determining the intracellular locations of fusion proteins. Fluorescently tagged, chromosomally expressed EpsC and EpsM form discrete foci along the lengths of the cells, different from the polar localization for green fluorescent protein (GFP)-EpsM previously described, as the fusions are balanced with all their interacting partner proteins within the T2S complex. Additionally, we observed that fluorescent foci in both chromosomal GFP-EpsC- and GFP-EpsM-expressing strains disperse upon deletion of epsD, suggesting that EpsD is critical to the localization of EpsC and EpsM and perhaps their assembly into the T2S complex.
authors.affiliation: Dept. of Microbiology and Immunology, University of Michigan Medical School, 1150 West Medical Center Drive, Ann Arbor, MI 48109, USA.
authors.forename: Suzanne R
authors.initials: SR
authors.lastname: Lybarger
authors.forename: Tanya L
authors.initials: TL
authors.lastname: Johnson
authors.forename: Miranda D
authors.initials: MD
authors.lastname: Gray
authors.forename: Aleksandra E
authors.initials: AE
authors.lastname: Sikora
authors.forename: Maria
authors.initials: M
authors.lastname: Sandkvist
mesh_terms: D019677:Artificial Gene Fusion; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry; D002772:Cholera Toxin / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014734:Vibrio cholerae / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; C406586:EpsD protein, Vibrio cholerae; C083759:EpsM protein, Vibrio cholerae; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D002772:Cholera Toxin