title: Determination of the Stoichiometry of the Complete Bacterial Type III Secretion Needle Complex Using a Combined Quantitative Proteomic Approach.
abstract: Precisely knowing the stoichiometry of their components is critical for investigating structure, assembly, and function of macromolecular machines. This has remained a technical challenge in particular for large, hydrophobic membrane-spanning protein complexes. Here, we determined the stoichiometry of a type III secretion system of Salmonella enterica serovar Typhimurium using two complementary protocols of gentle complex purification combined with peptide concatenated standard and synthetic stable isotope-labeled peptide-based mass spectrometry. Bacterial type III secretion systems are cell envelope-spanning effector protein-delivery machines essential for colonization and survival of many Gram-negative pathogens and symbionts. The membrane-embedded core unit of these secretion systems, termed the needle complex, is composed of a base that anchors the machinery to the inner and outer membranes, a hollow filament formed by inner rod and needle subunits that serves as conduit for substrate proteins, and a membrane-embedded export apparatus facilitating substrate translocation. Structural analyses have revealed the stoichiometry of the components of the base, but the stoichiometry of the essential hydrophobic export apparatus components and of the inner rod protein remain unknown. Here, we provide evidence that the export apparatus of type III secretion systems contains five SpaP, one SpaQ, one SpaR, and one SpaS. We confirmed that the previously suggested stoichiometry of nine InvA is valid for assembled needle complexes and describe a loose association of InvA with other needle complex components that may reflect its function. Furthermore, we present evidence that not more than six PrgJ form the inner rod of the needle complex. Providing this structural information will facilitate efforts to obtain an atomic view of type III secretion systems and foster our understanding of the function of these and related flagellar machines. Given that other virulence-associated bacterial secretion systems are similar in their overall buildup and complexity, the presented approach may also enable their stoichiometry elucidation.
authors.affiliation: From the ‡University of Tübingen, Interfaculty Institute of Microbiology and Infection Medicine (IMIT), Section of Cellular and Molecular Microbiology, Elfriede-Aulhorn-Str. 6, 72076 Tübingen, Germany;
authors.forename: Susann
authors.initials: S
authors.lastname: Zilkenat
authors.affiliation: §University of Tübingen, Proteome Center Tübingen, Auf der Morgenstelle 15, 72076 Tübingen, Germany;
authors.forename: Mirita
authors.initials: M
authors.lastname: Franz-Wachtel
authors.affiliation: ¶University of Tübingen, Center for Plant Molecular Biology (ZMBP), Auf der Morgenstelle 32, 72076 Tübingen, Germany;
authors.forename: York-Dieter
authors.initials: YD
authors.lastname: Stierhof
authors.affiliation: ‖Yale University School of Medicine, Department of Microbial Pathogenesis, 295 Congress Ave, New Haven, CT;
authors.forename: Jorge E
authors.initials: JE
authors.lastname: Galán
authors.affiliation: §University of Tübingen, Proteome Center Tübingen, Auf der Morgenstelle 15, 72076 Tübingen, Germany;
authors.forename: Boris
authors.initials: B
authors.lastname: Macek
authors.affiliation: From the ‡University of Tübingen, Interfaculty Institute of Microbiology and Infection Medicine (IMIT), Section of Cellular and Molecular Microbiology, Elfriede-Aulhorn-Str. 6, 72076 Tübingen, Germany; **German Center for Infection Research (DZIF), Partner-site Tübingen, Elfriede-Aulhorn-Str. 6, 72076 Tübingen, Germany samuel.wagner@med.uni-tuebingen.de.
authors.forename: Samuel
authors.initials: S
authors.lastname: Wagner
mesh_terms: D007553:Isotope Labeling / Q000379:methods; D013058:Mass Spectrometry; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry*; D055503:Protein Multimerization; D040901:Proteomics / Q000379:methods*; D012486:Salmonella typhimurium / Q000737:chemistry / Q000378:metabolism*; D000069296:Type III Secretion Systems / Q000737:chemistry*
chemical_list: D010455:Peptides; D000069296:Type III Secretion Systems