In bacterial type 3 secretion, substrate proteins are actively transported from the bacterial cytosol into the host cell cytosol by a large membrane-embedded machinery called the injectisome. Injectisomes transport secretion substrates in response to specific environmental signals, but the mechanisms by which the cytosolic secretion substrates and their chaperones are targeted to the type 3 secretion pathway remain unclear. Secretion activity and substrate selectivity are thought to be controlled by a sorting platform consisting of the proteins SctK, SctQ, SctL, and SctN, which together localize to the cytoplasmic side of membrane-embedded injectisomes. However, recent work revealed that sorting platform proteins also exhibit substantial cytosolic populations and that SctQ reversibly binds to and dissociates from the cytoplasmic side of membrane-embedded injectisomes. Based on these observations, we hypothesized that dynamic molecular turnover at the injectisome and cytosolic assembly among sorting platform proteins is a critical regulatory component of type 3 secretion. To determine whether assembled sorting platform complexes exist in the cytosol, we used 3D single-molecule tracking microscopy to measure the diffusive properties of sorting platform proteins in wild-type and mutant <i>Yersinia enterocolitica</i> cells. Our data show that both SctQ and SctL exist in distinct diffusive states in the cytosol, indicating that these proteins form stable homo- and hetero-oligomeric complexes in their native environment. These findings provide the first diffusive state-resolved insights into the dynamic regulatory network that interfaces stationary membrane-embedded injectisomes with the soluble cytosolic components of the type 3 secretion system.

Getting to the bottom of bacterial type 3 secretion by tracking biomolecular complexes in living cells

2019 Fall National Meeting

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