Development of roGFP2-derived redox probes for measurement of the glutathione redox potential in the cytosol of severely glutathione-deficient rml1 seedlings

Glutathione is important for detoxification, as a cofactor in biochemical reactions and as a thiol-redox buffer. The cytosolic glutathione buffer is normally highly reduced with glutathione redox potentials (E-GSH) of more negative than -310 mV. Maintenance of such negative redox potential is achieved through continuous reduction of glutathione disulfide by glutathione reductase (GR). Deviations from steady state glutathione redox homeostasis have been discussed as a possible mean to alter the activity of redox-sensitive proteins through switching of critical thiol residues. To better understand such signaling mechanisms it is essential to be able to measure E-GSH over a wide range from highly negative redox potentials down to potentials found in mutants that show already severe phenotypes. With the advent of redox-sensitive GFPs (roGFPs), understanding the in vivo dynamics of the thiol-based redox buffer system became within reach. The original roGFP versions, roGFP1 and roGFP2, however, have midpoint potentials between -280 and -290 mV rendering them fully oxidized in the ER and almost fully reduced in the cytosol, plastids, mitochondria, and peroxisomes. To extend the range of suitable probes we have engineered a roGFP2 derivative, roGFP2-iL, with a midpoint potential of about -238 mV. This value is within the range of redox potentials reported for homologous roGFP1-iX probes, albeit with different excitation properties. To allow rapid and specific equilibration with the glutathione pool, fusion constructs with human glutaredoxin 1 (GRX1) were generated and characterized in vitro. GRX1-roGFP2-iL proved to be suitable for in vivo redox potential measurements and extends the range of E-GSH values that can be measured in vivo with roGFP2-based probes from about -320 mV for GRX1-roGFP2 down to about -210 mV for GRX1-roGFP2-iL. Using both probes in the cytosol of severely glutathione-deficient rml1 seedlings revealed an E-GSH of about -260 mV in this mutant