Ratiometric fluorescence measurements of membrane potential generated by yeast plasma membrane H +-ATPase reconstituted into vesicles

Abstract

Potential-sensitive fluorescent probes oxonol V and oxonol VI were employed for monitoring membrane potential (Δ ψ) generated by the Schizosaccharomyces pombe plasma membrane H +-ATPase reconstituted into vesicles. Oxonol VI was used for quantitative measurements of the Δ ψ because its response to membrane potential changes can be easily calibrated, which is not possible with oxonol V. However, oxonol V has a superior sensitivity to Δ ψ at very low concentration of reconstituted vesicles, and thus it is useful for testing quality of the reconstitution. Oxonol VI was found to be a good emission-ratiometric probe. We have shown that the reconstituted H +-ATPase generates Δ ψ of about 160 mV on the vesicle membrane. The generated Δ ψ was stable at least over tens of minutes. An influence of the H + membrane permeability on the Δ ψ buildup was demonstrated by manipulating the H + permeability with the protonophore CCCP. Ratiometric measurements with oxonol VI thus offer a promising tool for studying processes accompanying the yeast plasma membrane H +-ATPase-mediated Δ ψ buildup.