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 envelopespanning 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. Molecular & Cellular

Molecular Models for the Core Components of the Flagellar Type-III Secretion Complex

“…While it is possible that this region may face a solvent accessible pore, the mutation studies mentioned above suggest that this collection of charged residues may constitute the proton channel that drives secretion. Although FlhA is the most likely candidate for the proton translocation function, being the most abundant protein in the export gate [61] and essential for secretion [59, 62], a complete transmembrane proton conduction pathway is not obvious in our model.…”

Visualization and characterization of individual type III protein secretion machines in live bacteria

“…Although the composition and stoichiometry of the needle complex components are known (16,18,45), less information is available regarding the cytoplasmic sorting platform, and the inability to isolate this substructure has hampered efforts to determine its stoichiometry (20). To gain insights into the sorting platform, we constructed a S. Typhimurium strain that expresses a core component of this substructure, SpaO, tagged with mEos3.2 from its native chromosomal context.…”

Stoichiometry determination of macromolecular membrane protein complexes

“…To determine the ratios of transmembrane complex proteins, stable isotope-labeled standard mass spectrometry based approaches are promising as ratios of all complex components can be analyzed in one multiplex run. We were able to obtain stoichiometries ranging from 1 to 24 for a 3 MDa complex, using an AQUA-complemented PCS strategy (Zilkenat et al, 2016). Exploiting the recently introduced hierarchical standards may further improve both range and error margins of this approach and thus may facilitate the robust and accurate stoichiometry determination of large membrane-spanning protein complexes.…”

“…Since flanking regions influence the efficiency of tryptic digestion, this trick results in a more similar digestion behavior of sample and standard, in particular for transmembrane complexes (Kito and Ito, 2008). We were able to use the PCS strategy to determine the stoichiometry of the complete needle complex of a type III secretion system of Salmonella Typhimurium, a multi-MDa transmembrane complex with a stoichiometric range of 1-24 (disregarding the needle filament itself) (Zilkenat et al, 2016). Knowledge of the stoichiometry of three of its components (Schraidt and Marlovits, 2011) allowed the calculation of absolute numbers per complex from protein : protein ratios obtained by the PCS approach.…”

“…Cryo electron microscopy (10 Å resolution); X-ray crystallography (C-terminal domain of export apparatus protein); Stable isotope-labeled standards mass spectrometry; Photobleaching (translocator) (Worrall et al, 2010;Schraidt and Marlovits, 2011;Romano et al, 2016;Zilkenat et al, 2016) Cellulose synthesis complex > 5 3 18 Plants Cryo electron microscopy; Quantitative immunoblotting (CESA4, 7 and 8); Photobleaching (CESA3) (Kimura et al, 1999;Chen et al, 2014;Hill et al, 2014) Nuclear pore complex…”