Membrane voltage-dependent activation mechanism of the bacterial flagellar protein export apparatus

Keiichi Namba,Tohru Minamino,Yusuke V Morimoto,Miki Kinoshita

doi:10.1073/pnas.2026587118

Abstract

The proton motive force (PMF) consists of the electric potential difference (Δψ), which is measured as membrane voltage, and the proton concentration difference (ΔpH) across the cytoplasmic membrane. The flagellar protein export machinery is composed of a PMF-driven transmembrane export gate complex and a cytoplasmic ATPase ring complex consisting of FliH, FliI, and FliJ. ATP hydrolysis by the FliI ATPase activates the export gate complex to become an active protein transporter utilizing Δψ to drive proton-coupled protein export. An interaction between FliJ and a transmembrane ion channel protein, FlhA, is a critical step for Δψ-driven protein export. To clarify how Δψ is utilized for flagellar protein export, we analyzed the export properties of the export gate complex in the absence of FliH and FliI. The protein transport activity of the export gate complex was very low at external pH 7.0 but increased significantly with an increase in Δψ by an upward shift of external pH from 7.0 to 8.5. This observation suggests that the export gate complex is equipped with a voltage-gated mechanism. An increase in the cytoplasmic level of FliJ and a gain-of-function mutation in FlhA significantly reduced the Δψ dependency of flagellar protein export by the export gate complex. However, deletion of FliJ decreased Δψ-dependent protein export significantly. We propose that Δψ is required for efficient interaction between FliJ and FlhA to open the FlhA ion channel to conduct protons to drive flagellar protein export in a Δψ-dependent manner.

Highlights

| | | bacterial flagellum membrane voltage proton motive force | type III protein export Salmonella through the channel into the force for high-speed rotation of the long helical filament [3, 4]
We show that an increase in Δψ generated by an upward shift of the external pH from 7.0 to 8.5 activates flagellar protein export by this mutant even in the absence of ΔpNa, suggesting the presence of a Δψ-dependent activation mechanism for protoncoupled protein secretion by the export gate complex
Because the SMF was larger than the proton motive force (PMF) under our experimental conditions (SI Appendix, Table S4), we propose that the increased Δψ acts on the FlhA ion channel to facilitate the inward-directed flow of both H+ and Na+ when they are coupled to outward-directed protein translocation

Summary

Introduction

| | | bacterial flagellum membrane voltage proton motive force | type III protein export Salmonella through the channel into the force for high-speed rotation of the long helical filament [3, 4]. The flagellar protein transporter consists of a PMF-driven export gate complex made of five transmembrane proteins, FlhA, FlhB, FliP, FliQ, and FliR, and an ATPase ring complex consisting of three cytoplasmic proteins, FliH, FliI, and FliJ (SI Appendix, Fig. S1) [5, 6]. These proteins are evolutionarily related to those of the virulence-associated type III secretion systems of pathogenic bacteria, which inject effector proteins into eukaryotic host cells for invasion [7]. The IMF is the sum of the electrical (Δψ) and chemical (ΔpI) potential differences of ions such as protons (H+) (the proton motive force [PMF]) and sodium ions (Na+) (the sodium motive force [SMF]) across the membrane and is defined by Eq 1: IMF

Methods

Results

Conclusion