Life science
Medical & healthcare, Drug development

Membrane voltage-dependent activation mechanism of thebacterial flagellar protein export apparatus
Graduate school of Frontier Biosciences
Associate Professor Tohru Minamino
https://researchmap.jp/tohru_minamino?lang=en

Life science

Abstract
Proton motive force (PMF) across the cytoplasmic membraneconsists of the electric potential difference and the proton concentration difference. Many motile bacteria employ the flagellar typeIII secretion system (fT3SS) to construct a supramolecular motility
machine, the flagellum, on the cell surface. The fT3SS is composedof a transmembrane export gate complex powered by the PMF anda cytoplasmic ATPase ring complex. If the ATPase ring complex
does not work properly under a given environmental condition, theexport gate complex remains inactive. However, when membrane
voltage exceeds a certain threshold value, the export gate complex becomes an active protein transporter to drive proton-coupledprotein export. This observation suggests that the export gate complex has a voltage-gated activation mechanism.

▍ Background & Results
The fT3SS consists of a PMF-driven transmembrane export gatecomplex made of FlhA, FlhB, FliP, FliQ, and FliR and a cytoplasmic
ATPase ring complex consisting of FliH, FliI, and FliJ. ATP hydrolysis by the FliI ATPase activates the transmembrane export gate
complex through an interaction between FliJ and FlhA, allowing
the export gate complex to utilize the electric potential difference,
which is measured as membrane voltage, to couple the proton flow
through the FlhA proton channel with protein translocation througha polypeptide channel formed by FliP, FliQ, and FliR. However, itremains unknown how membrane voltage is utilized for flagellar
protein export. One Salmonella mutant with FliI that cannot work as
a flagellum-specific ATPase in the fT3SS showed no visible flagellar
filaments at an external pH value of 7.0. However, when this mutant
strain was cultured at an external pH value of 8.5, many cells produced a few flagella. Because an upward shift of external pH from
7.0 to 8.5 increased membrane voltage significantly but decreased
total PMF in Salmonella cells, our observations strongly suggestthat the protein transport activity of the export gate complex isquite low at external pH 7.0 but increases markedly with increasing
membrane voltage. Furthermore, an increase in membrane voltage
significantly stabilized the FlhA-FliJ interaction, allowing FlhA toopen its proton channel to conduct protons to be coupled with flagellar protein export.

Figure 1. Electron microgram of Salmonella cell and schematic diagram of thebacterial flagellar type III protein export apparatus, which is located at the baseof the flagellum.

Figure 2. Capability of the Salmonella mutant with FliI, which cannot work as a flagellum-specific ATPase in the flagellar type III protein export apparatus, to form flagella.

▍ Significance of the research and Future perspective
In addition to clarifying how membrane voltage is used for flagellar protein export, this is the first report describing a membrane
voltage-dependent activation mechanism used for a biological
function other than voltage-gated ion channels. These discoveries
are expected to enable drug screening for the treatment of infectious diseases to replace conventional antibiotics by directly targeting the injectisome of pathogenic bacteria, which is functionallyand structurally identical to the fT3SS.

P a t e n t
Minamino, Tohru; Morimoto, Yusuke; Kinoshita, Miki et al. Membrane voltage-dependent activation mechanism of the bacterial flagellar
T r e a t i s e protein export apparatus. Proceedings of the National Academy of Sciences of the United States of America. 2021, 118 (22), e2026587118,
doi: 10.1073/pnas.2026587118
UR
L https://www.fbs.osaka-u.ac.jp/ja/research_results/papers/detail/1022
K e y w o r d bacterial flagella, type III secretion system, membrane voltage, bacterial infection