Determination of the Stoichiometry of the Complete Bacterial Type III Secretion Needle Complex Using a Combined Quantitative Proteomic Approach

• DOI: 10.1074/mcp.m115.056598
• Publication Date: 2016-02-22T01:08:07Z
• Authors: Susann Zilkenat; Mirita Franz-Wachtel; York-Dieter Stierhof; Jorge E. Galán; Boris Macek; Samuel Wagner
• Keywords: Quantitative Analysis; Stoichiometry

Precisely knowing the stoichiometry of their components is critical for investigating structure, assembly, and function macromolecular machines. This has remained a technical challenge in particular large, hydrophobic membrane-spanning protein complexes. Here, we determined type III secretion system Salmonella enterica serovar Typhimurium using two complementary protocols gentle complex purification combined with peptide concatenated standard synthetic stable isotope-labeled peptide-based mass spectrometry. Bacterial systems are cell envelope-spanning effector protein-delivery machines essential colonization survival many Gram-negative pathogens symbionts. The membrane-embedded core unit these systems, termed needle complex, composed base that anchors machinery to inner outer membranes, hollow filament formed by rod subunits serves as conduit substrate proteins, export apparatus facilitating translocation. Structural analyses have revealed base, but remain unknown. provide evidence contains five SpaP, one SpaQ, SpaR, SpaS. We confirmed previously suggested nine InvA valid assembled complexes describe loose association other may reflect its function. Furthermore, present not more than six PrgJ form complex. Providing this structural information will facilitate efforts obtain an atomic view foster our understanding related flagellar Given virulence-associated bacterial similar overall buildup complexity, presented approach also enable elucidation. Type (T3SS), evolutionary structurally flagella (1Abby S.S. Rocha E.P.C. non-flagellar evolved from flagellum diversified into host-cell adapted systems.PLoS Genet. 2012; 8: e1002983Crossref PubMed Scopus (186) Google Scholar), used pathogenic or symbiotic bacteria inject proteins eukaryotic host cells order promote (2Galán J.E. Lara-Tejero M. Marlovits T.C. Wagner S. systems: specialized nanomachines delivery target cells.Annu. Rev. 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