14 research outputs found
Genetic Characterization of Conserved Charged Residues in the Bacterial Flagellar Type III Export Protein FlhA
For assembly of the bacterial flagellum, most of flagellar proteins are transported to the distal end of the flagellum by the flagellar type III protein export apparatus powered by proton motive force (PMF) across the cytoplasmic membrane. FlhA is an integral membrane protein of the export apparatus and is involved in an early stage of the export process along with three soluble proteins, FliH, FliI, and FliJ, but the energy coupling mechanism remains unknown. Here, we carried out site-directed mutagenesis of eight, highly conserved charged residues in putative juxta- and trans-membrane helices of FlhA. Only Asp-208 was an essential acidic residue. Most of the FlhA substitutions were tolerated, but resulted in loss-of-function in the ÎfliH-fliI mutant background, even with the second-site flhB(P28T) mutation that increases the probability of flagellar protein export in the absence of FliH and FliI. The addition of FliH and FliI allowed the D45A, R85A, R94K and R270A mutant proteins to work even in the presence of the flhB(P28T) mutation. Suppressor analysis of a flhA(K203W) mutation showed an interaction between FlhA and FliR. Taken all together, we suggest that Asp-208 is directly involved in PMF-driven protein export and that the cooperative interactions of FlhA with FlhB, FliH, FliI, and FliR drive the translocation of export substrate
An energy transduction mechanism used in bacterial flagellar type III protein export
Flagellar proteins of bacteria are exported by a specific export apparatus. FliI ATPase forms a complex with FliH and FliJ and escorts export substrates from the cytoplasm to the export gate complex, which is made up of six membrane proteins. The export gate complex utilizes proton motive force across the cytoplasmic membrane for protein translocation, but the mechanism remains unknown. Here we show that the export gate complex by itself is a protonâprotein antiporter that uses the two components of proton motive force, ÎÏ and ÎpH, for different steps of the protein export process. However, in the presence of FliH, FliI and FliJ, a specific binding of FliJ with an export gate membrane protein, FlhA, is brought about by the FliHâFliI complex, which turns the export gate into a highly efficient, ÎÏ-driven protein export apparatus
Export Mechanisms and Energy Transduction in Type-III Secretion Machines
International audienc
A dynamic and adaptive network of cytosolic interactions governs protein export by the T3SS injectisome
Many bacteria use a type III secretion system to inject effector proteins into host cells. Selection
and export of the effectors is controlled by a set of soluble proteins at the cytosolic interface of
the membrane spanning type III secretion âinjectisomeâ. Combining fluorescence microscopy,
biochemical interaction studies and fluorescence correlation spectroscopy, we show that in live
Yersinia enterocolitica bacteria these soluble proteins form complexes both at the injectisome and
in the cytosol. Binding to the injectisome stabilizes these cytosolic complexes, whereas the free
cytosolic complexes, which include the type III secretion ATPase, constitute a highly dynamic
and adaptive network. The extracellular calcium concentration, which triggers activation of the
T3SS, directly influences the cytosolic complexes, possibly through the essential component
SctK/YscK, revealing a potential mechanism involved in the regulation of type III secretion