The bacterial flagellar type III secretion system (fT3SS) exports structural subunits required for flagellar assembly by coupling protein translocation to ion motive force across the cytoplasmic membrane. Efficient activation of the transmembrane export gate depends on a cytoplasmic ATPase complex composed of FliH, FliI, and FliJ, which are also involved in substrate delivery. However, how these proteins mechanistically integrate substrate delivery with gate activation remains unclear. Here, we uncoupled these two functions by cross-complementation analyses using ATPase components from the Na+-driven polar flagellum of Vibrio and the H+-driven flagellum of Salmonella. Despite low sequence identity, Vibrio FliJ complemented a Salmonella ΔfliJ mutant and restored Na+-independent protein export to a substantial extent, demonstrating a highly conserved mechanism of export gate activation. In contrast, Vibrio FliH and FliI exhibited interspecies incompatibility when expressed individually, and their co-expression in a Salmonella ΔfliH-fliI mutant supported protein export only under Na+-coupled conditions, consistent with the failure to activate the H+-driven export gate. Biochemical analyses revealed species-specific interactions between FliH and FliI, while high-speed atomic force microscopy showed that the Vibrio FliH-FliI complex retains the ability to assemble into ring-shaped structures. Together, these findings demonstrate that ATPase ring assembly and substrate delivery are mechanistically separable from export gate activation, revealing distinct and differentially conserved roles of the flagellar ATPase complex in coupling ATP hydrolysis to ion-driven protein export.
Minamino et al. (Fri,) studied this question.