40Many medically relevant Gram-negative bacteria use the type III secretion system (T3SS) to 41 translocate effector proteins into the host for their invasion and intracellular survival. A multi-42 protein complex located at the cytosolic interface of the T3SS is proposed to act as a sorting 43 platform by selecting and targeting substrates for secretion through the system. However, the 44 precise stoichiometry and 3D organization of the sorting platform components is unknown. Here 45 we reconstitute soluble complexes of the Salmonella Typhimurium sorting platform proteins 46 including the ATPase InvC, the regulator OrgB, the protein SpaO and a recently identified 47 subunit SpaOC, which we show to be essential for the solubility of SpaO. We establish domain-48 domain interactions, determine for the first time the stoichiometry of each subunit within the 49 complexes by native mass spectrometry and gain insight into their organization using small-angle 50 X-ray scattering. Importantly, we find that in solution the assembly of SpaO/SpaOC/OrgB/InvC 51 adopts an extended L-shaped conformation resembling the sorting platform pods seen in in situ 52 cryo-electron tomography, proposing that this complex is the core building block that can be 53 conceivably assembled into higher oligomers to form the T3SS sorting platform. The determined 54 molecular arrangements of the soluble complexes of the sorting platform provide important 55 insights into its architecture and assembly. 56 refer to as SpaOC (16,17). This short product interacts with the full-length protein in other species 87 and thus could represent an additional structural component of the sorting platform (12,(18)(19)(20). 88 However, the function of SpaOC in type III secretion is elusive, and how it interacts with the 89 other subunits of the sorting platform is unknown. Moreover, the precise protein composition and 90 spatial molecular organization of the sorting platform, as well its assembly process and 91 mechanism of action in substrate sorting remain uncertain.
92In this study, we reconstitute and analyze for the first time the soluble assembling units of the 93 Salmonella Typhimurium SPI-1 sorting platform using purified proteins. We observe that SpaOC, 94 the second protein product of the gene spaO, is required for fully efficient type III function and 95 for the stability of the sorting platform complexes in solution. Using native mass spectrometry 96 (MS), small-angle X-ray scattering (SAXS) and multi-angle light scattering (MALS), we 97 characterize different substructures of the sorting platform, determining their stoichiometry and 98 association into SpaO/SpaOC/OrgB/InvC complexes. These complexes adopt an extended L-99 shaped conformation in solution that mirrors a segment of the sorting platform visualized by 100 CET. Our data present the most detailed assembly of the Salmonella Typhimurium SPI-1 sorting 101 platform in solution, reporting the conformation of what we propose is the core building block to 102 assemble the sorting platform at t...