title: Francisella tularensis RipA protein topology and identification of functional domains.
abstract: Francisella tularensis is a Gram-negative coccobacillus and is the etiological agent of the disease tularemia. Expression of the cytoplasmic membrane protein RipA is required for Francisella replication within macrophages and other cell types; however, the function of this protein remains unknown. RipA is conserved among all sequenced Francisella species, and RipA-like proteins are present in a number of individual strains of a wide variety of species scattered throughout the prokaryotic kingdom. Cross-linking studies revealed that RipA forms homoligomers. Using a panel of RipA-green fluorescent protein and RipA-PhoA fusion constructs, we determined that RipA has a unique topology within the cytoplasmic membrane, with the N and C termini in the cytoplasm and periplasm, respectively. RipA has two significant cytoplasmic domains, one composed roughly of amino acids 1 to 50 and the second flanked by the second and third transmembrane domains and comprising amino acids 104 to 152. RipA functional domains were identified by measuring the effects of deletion mutations, amino acid substitution mutations, and spontaneously arising intragenic suppressor mutations on intracellular replication, induction of interleukin-1β (IL-1β) secretion by infected macrophages, and oligomer formation. Results from these experiments demonstrated that each of the cytoplasmic domains and specific amino acids within these domains are required for RipA function.
authors.affiliation: Department of Microbiology and Immunology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
authors.forename: Brittany L
authors.initials: BL
authors.lastname: Mortensen
authors.forename: James R
authors.initials: JR
authors.lastname: Fuller
authors.forename: Sharon
authors.initials: S
authors.lastname: Taft-Benz
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Collins
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Kawula
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D005604:Francisella tularensis / Q000737:chemistry* / Q000254:growth & development / Q000378:metabolism* / Q000472:pathogenicity; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D053583:Interleukin-1beta / Q000378:metabolism; D008264:Macrophages / Q000276:immunology / Q000382:microbiology; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019897:Periplasm / Q000737:chemistry; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D013194:Staining and Labeling / Q000379:methods; D013489:Suppression, Genetic
chemical_list: D001426:Bacterial Proteins; D053583:Interleukin-1beta; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D010744:Phosphoric Monoester Hydrolases