In Vitro Demonstration of Light-Driven Na+/H+ Pumping by a Microbial Rhodopsin

Abstract

A new subfamily of rhodopsins was discovered in bacteria and proposed to function as dual function light-driven H+/Na+ pumps, ejecting Na+ from cells in the presence of Na+ and H+ in its absence (Inoue et al 2013. Nat. Commun. 4:1678). This proposal was based primarily on light-induced proton flux measurements in suspensions of E. coli cells expressing the pigments. However, because E. coli cells contain numerous proteins that mediate proton fluxes, indirect effects on proton movements involving endogenous bioenergetics components could not be excluded. Therefore, an in vitro system consisting of the purified pigment in the absence of other proteins was needed to assign the Na+ and H+ transport definitively. We expressed IAR from Indibacter alkaliphilus in E. coli cells and observed similar ion fluxes as reported for KR2 from Dokdonia eikasta reported earlier. We purified and reconstituted IAR into large unilamellar vesicles (LUVs), and demonstrated the proton flux criteria of light-dependent electrogenic Na+ pumping activity in vitro, namely Na+-dependent light-induced passive proton flux enhanced by protonophore. The proton flux was out of the LUV lumen, increasing lumenal pH. In contrast, illumination of the LUVs in a Na+-free suspension medium caused a decrease of lumenal pH, eliminated by protonophore, showing H+ pumping activity. The direction of proton fluxes indicated that IAR was inserted inside-out into the sealed LUV system, which we confirmed by site-directed spin-label EPR spectroscopy. The in vitro LUV system proves that the dual light-driven H+/Na+ pumping function of IAR is intrinsic to the single rhodopsin protein and enables study of the transport activities without perturbation by bioenergetics ion fluxes encountered in vivo. We are currently investigating ion fluxes through natural anion channelrhodopsins with this system.