Distinct cytosolic complexes containing the type III secretion system ATPase resolved by 3D single-molecule tracking in live Yersinia enterocolitica

The membrane-embedded injectisome, the structural component of the virulence-associated type III secretion system (T3SS), is used by gram-negative bacterial pathogens to inject species-specific effector proteins into eukaryotic host cells. The cytosolic injectisome proteins are required for export of effectors and display both stationary, injectisome-bound populations as well as freely-diffusing cytosolic populations. How the cytosolic injectisome proteins interact with each other in the cytosol and associate with membrane-embedded injectisomes remains unclear. Here, we utilize 3D single-molecule tracking to resolve distinct cytosolic complexes of injectisome proteins in living Yersinia enterocolitica cells. Tracking of the eYFP-labeled ATPase, Ye SctN, and its regulator, Ye SctL, reveals that these proteins form a cytosolic complex with each other and then further with Ye SctQ. Ye SctNL and Ye SctNLQ complexes can be observed both in wild type cells and in Δ sctD mutants, which cannot assemble injectisomes. In Δ sctQ mutants, the relative abundance of the Ye SctNL complex is considerably increased. These data indicate that distinct cytosolic complexes of injectisome proteins can form prior to injectisome binding, which has important implications for how injectisomes are functionally regulated. Importance Injectisomes are membrane-embedded, multiprotein assemblies used by bacterial pathogens to inject virulent effector proteins into eukaryotic host cells. Protein secretion is regulated by cytosolic proteins that dynamically bind and unbind at injectisomes. However, how these regulatory proteins interact with each other remains unknown. By measuring the diffusion rates of single molecules in living cells, we show that cytosolic injectisome proteins form distinct oligomeric complexes with each other prior to binding to injectisomes. We additionally identify the molecular compositions of these complexes and quantify their relative abundances. Quantifying to what extent cytosolic proteins exist as part of larger complexes in living cells has important implications for deciphering the complexity of biomolecular mechanisms. The results and methods reported here are thus relevant for advancing our understanding of how injectisomes, and related multiprotein assemblies, such as bacterial flagellar motors, are functionally regulated. ### Competing Interest Statement The authors have declared no competing interest.