Abstract
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 (EGSH) 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 EGSH 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 EGSH 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 EGSH of about −260 mV in this mutant.
Highlights
Thiol redox biochemistry is considered to play a fundamental role in cellular processes including signaling and cell fate decisions
RoGFP2-iL was fused behind human glutaredoxin 1 (GRX1) to generate GRX1-roGFP2-iL (Figure 1C) to ensure specificity of the novel probe for EGSH similar to other redox-sensitive GFPs (roGFPs) variants used before (Gutscher et al, 2008; Meyer and Dick, 2010; Albrecht et al, 2013; Birk et al, 2013)
Peroxisomes, mitochondria, and plastids in Arabidopsis wild-type plants maintain a highly reduced glutathione buffer with redox potentials of less than −310 mV, the endoplasmic reticulum (ER) is far less reducing with EGSH values of less than −240 mV (Meyer et al, 2007; Schwarzländer et al, 2008)
Summary
Thiol redox biochemistry is considered to play a fundamental role in cellular processes including signaling and cell fate decisions. Current variants of roGFPs are largely inadequate in mutants with very low glutathione levels, generally oxidizing conditions like in the ER, or oxidizing conditions triggered by pathological processes. These limitations demand the development of further roGFP variants with less negative midpoint potentials. Glutathione constitutes one of the most important redox buffer systems in the cell. EGSH depends on the absolute glutathione concentration and the ratio www.frontiersin.org
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