Determination of Local pH Differences within Living Cells by High-resolution pH Imaging Based on pH-sensitive GFP Derivative, pHluorin(M153R).

The bacterial flagellar type III protein export apparatus is composed of a transmembrane export gate complex and a cytoplasmic ATPase complex. The export apparatus requires ATP hydrolysis and the proton motive force across the cytoplasmic membrane to unfold and transport flagellar component proteins for the construction of the bacterial flagellum (Minamino, 2014). The export apparatus is a proton/protein antiporter that couples the proton flow with protein transport through the gate complex ( Minamino , 2011 ). A transmembrane export gate protein, FlhA, acts as an energy transducer along with the cytoplasmic ATPase complex ( Minamino , 2016 ). To directly measure the proton flow through the FlhA channel that is coupled with the flagellar protein export, we have developed an pH imaging system with high spatial and pH resolution ( Morimoto , 2016 ). Here, we describe how we measure the local pH near the export apparatus in living cells ( Morimoto , 2016 ). Our approach can be applied to a wide range of living cells. Because local pH is one of the most important parameters to monitor cellular activities of living cells, our protocol would be widely used for diverse areas of life sciences.