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.
Highlights
From the ‡University of Tubingen, Interfaculty Institute of Microbiology and Infection Medicine (IMIT), Section of Cellular and Molecular Microbiology, Elfriede-Aulhorn-Str. 6, 72076 Tubingen, Germany; §University of Tubingen, Proteome Center Tubingen, Auf der Morgenstelle 15, 72076 Tubingen, Germany; ¶University of Tubingen, Center for Plant Molecular Biology (ZMBP), Auf der Morgenstelle 32, 72076 Tubingen, Germany; ʈYale University School of Medicine, Department of Microbial Pathogenesis, 295 Congress Ave, New Haven, CT; **German Center for Infection Research (DZIF), Partner-site Tubingen, Elfriede-Aulhorn-Str. 6, 72076 Tubingen, Germany
It consists of a base that anchors the complex in the bacterial inner and outer membranes [3], an inner membrane-embedded export apparatus facilitating substrate translocation located at the center of the base [4], and a filamentous inner rod and needle, which protrude from the bacterial surface and serve as conduit for substrates [2] (Fig. 1)
By combining gentle membrane protein complex purification and quantitative mass spectrometry, we show that MS-based stoichiometry determination can be extended to investigating highly hydrophobic complexes of a wide stoichiometric range such as multiple other bacterial protein secretion machines
Summary
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. The low-resolution structural analysis of isolated needle complexes revealed a stoichiometry of 12–15 copies for the outer membrane secretin ring of T3SSs of different bacteria and of 12–24 copies for the two inner membrane ring proteins [11,12,13,14]. Only the sizeable cytoplasmic domains of several homologs of the hydrophobic export apparatus components SpaS and InvA were amenable to high-resolution structural studies (16 –24). Besides SpaS, the stoichiometry of the minor export apparatus components SpaP, SpaQ, and SpaR is unknown
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