Local redox environment beneath biological membranes probed by palmitoylated-roGFP

Production of reactive oxygen species (ROS) and consequent glutathione oxidation are associated with various physiological processes and diseases, including cell differentiation, senescence, and inflammation. GFP-based redox sensors provide a straight-forward approach to monitor ROS levels and glutathione oxidation within a living cell at the subcellular resolution. We utilized palmitoylated versions of cytosolic glutathione and hydrogen peroxide sensors (Grx1-roGFP2 and roGFP2-Orp1, respectively) and demonstrated a unique redox environment near biological membranes. In HeLa cells, cytosolic glutathione was practically completely reduced (E_GSH/GSSG = - 333mV) and hydrogen peroxide level was under the detectable range. In contrast, the cytoplasmic milieu near membranes of intracellular vesicles exhibited significant glutathione oxidation (E_GSH/GSSG > - 256mV) and relatively high H₂O₂ production, which was not observed for the plasma membrane. These vesicles colocalized with internalized EGFR, suggesting that H₂O₂ production and glutathione oxidation are characteristics of cytoplasmic surfaces of the endocytosed vesicles. The results visually illustrate local redox heterogeneity within the cytosol for the first time.