An optogenetic assay method for electrogenic transporters using Escherichia coli co-expressing light-driven proton pump

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Abstract

In organisms, nutrients and wastes move across the cellular membrane, in which membrane-embedded transporters facilitate and inhibit the movement. Despite the physiological significances, the currently used assay methods for transporter activities require tedious preparation and analytical processes. In this study, we report the isotope-free and label-free measurement system for the transport activities of electrogenic transporters. In the system, two molecules, a light-driven inward proton pump rhodopsin, xenorhodopsin (XeR), and a representative of an electrogenic transporter, an oxalate transporter (OxlT), were co-expressed in Escherichia coli cells. The light illumination of the cells co-expressing XeR and OxlT showed an increase in the pH of the bulk solution and that the extent of the pH change is significantly enhanced by adding the oxalate, suggesting the light-induced inward proton transport by XeR coupled to the negative electrogenic transport by OxlT. Such a pH increase was dependent on the oxalate concentration, but not on the XeR expression level. Of note, pH increase was not observed for the nonfunctional mutants of OxlT, R272A, and K355Q, supporting the validity of the system. Thus, we successfully developed an optogenetic assay method for electrogenic transporters using E. coli co-expressing light-driven proton pump.

Original languageEnglish
Pages (from-to)2161-2169
Number of pages9
JournalProtein Science
Volume30
Issue number10
DOIs
Publication statusPublished - Oct 2021

Keywords

  • E. coli
  • electrogenic
  • light-driven proton pump
  • oxalate transporter
  • transporter
  • xenorhodopsin

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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