Single-molecule tracking in liveYersinia enterocoliticareveals distinct cytosolic complexes of injectisome subunits

Abstract

In bacterial type 3 secretion, substrate proteins are actively transported from the bacterial cytoplasm into the host cell cytoplasm by a large membrane-embedded machinery called the injectisome. Injectisomes transport secretion substrates in response to specific environmental signals, but the molecular details by which the cytosolic secretion substrates are selected and transported through 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 additionally 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 sorting platform complexes exist in the cytosol, we measured the diffusive properties of the two central sorting platform proteins, SctQ and SctL, using live cell high-throughput 3D single-molecule tracking microscopy. Single-molecule trajectories, measured in wild-type and mutant Yersinia enterocolitica cells, reveal that both SctQ and SctL exist in several distinct diffusive states in the cytosol, indicating that these proteins form stable homo- and hetero-oligomeric complexes in their native environment. Our 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.

Dynamic relocalization of cytosolic type III secretion system components prevents premature protein secretion at low external pH

“…In the cytosol, SctK/Q/L/N form a dynamic adaptive network, with a variety of complexes of different stoichiometries that change their composition in response to different external conditions 18 , 25 . Strikingly, protein interactions and exchange rates amongst the cytosolic components correlate with the activity of the injectisome 18 , 24 , 26 . However, the precise role of the cytosolic components in the secretion process remains unclear to this date.…”

Dynamics of Bacterial Signal Recognition Particle at a Single Molecule Level

“…translating fraction, or if a putative SRP/FtsY/ribosome NC complex can form within the cytosol. We generated speed maps of Ffh, FtsY, and L1, considering the apparent diffusion D * (Rocha et al, 2018;Banaz et al, 2019) obtained from the linear fit of MSD curves on the first 4 time points (t = 1. . .,4• t).…”

Adaptivity and dynamics in type III secretion systems

“…A subsequent PALM analysis in S PI‐1 noted a similar cellular distribution for the sorting platform components (Zhang et al, 2017), indicating that the presence of cytosolic sorting platform proteins and subunit exchange is a universal feature of injectisomes. Fluorescence correlation spectroscopy (FCS) (Diepold et al, 2017) and high‐throughput single‐molecule tracking (Rocha et al, 2018) revealed distinct subcomplexes of the cytosolic T3SS components with different stoichiometries. A combined small angle X‐ray scattering (SAXS)/quantitative mass spec analysis characterized several of these complexes, with the most prominent having a stoichiometry of SctQ‐Qc 2 ‐L 2 ‐N and (SctQ‐Qc 2 ) 2 ‐L 2 ‐N, respectively (Bernal et al, 2019).…”