Redox-sensitive YFP sensors monitor dynamic nuclear and cytosolic glutathione redox changes

Abstract

Intracellular redox homeostasis is crucial for many cellular functions but accurate measurements of cellular compartment-specific redox states remain technically challenging. To better characterize redox control in the nucleus, we targeted a yellow fluorescent protein-based redox sensor (rxYFP) to the nucleus of the yeast S. cerevisiae. Parallel analyses of the redox state of nucleus-rxYFP and cytosol-rxYFP allow us to monitor distinctively dynamic glutathione (GSH) redox changes within these two compartments in a given condition. We observed that the nuclear GSH redox environment is highly reducing and similar to the cytosol under steady state conditions. Furthermore, these sensors are able to detect redox variations specific for their respective compartments in glutathione reductase (Glr1) and thioredoxin pathway (Trr1, Trx1, Trx2) mutants that have altered subcellular redox environments. Our mutant redox data provide in vivo evidence that glutathione and the thioredoxin redox system play distinct but overlapping functions in controlling subcellular redox environments. We also monitored the dynamic response of nucleus-rxYFP and cytosol-rxYFP to GSH depletion and to exogenous low and high doses of H2O2 bursts. These observations indicate a rapid and almost simultaneous oxidation of both nucleus-rxYFP and cytosol-rxYFP, highlighting the robustness of the rxYFP sensors in measuring real-time compartmental redox changes. Taken together, our data suggest that the highly reduced yeast nuclear and cytosolic redox states are maintained independently to some extent and under distinct but subtle redox regulation. Nucleus- and cytosol- rxYFP register compartment-specific localized redox fluctuations that may involve exchange of reduced and/or oxidized glutathione between these two compartments. Finally, we confirmed that GSH depletion has profound effects on mitochondrial genome stability but little effect on nuclear genome stability, thereby emphasizing that the critical requirement for GSH during growth is linked to a mitochondria-dependent process.

Intracellular reduction/activation of a disulfide switch in thiosemicarbazone iron chelators

“…As genetically encoded biosensors continue to provide new information on the compartmentalization of intracellular redox potentials, 31, 32 it is likely that more details on the extent and location of intracellular disulfide reductions will become available. In this simplified model of intracellular conditions containing micromolar chelation system and millimolar glutathione buffer, the chelator after equilibration was found prevalently in its reduced iron-binding form at glutathione half-cell potentials that have been associated to proliferating and malignant cells ( E hc ≤ −220 mV vs SHE).…”

Functions and Cellular Compartmentation of the Thioredoxin and Glutathione Pathways in Yeast

“…These differences may also be explained by a Dtrr1 gain of function caused by toxic accumulation of oxidized TRX leading to disulfide stress, as shown in E. coli (89). The latter hypothesis is supported by improvement of both growth and peroxide tolerance phenotypes by further deletion of TRX1 and TRX2 (Dtrr1Dtrx1Dtrx2) (17,94,105). Anyhow, lack of the cell cycle defect and organic sulfur auxotrophy of Dtrr1 also indicates that enough TRX activity remains in the cell to reduce RNR and PAPS reductase.…”

“…Such an assumption is borne out by the observation that the GSH redox potential (E GSH ) is similar in the cytosol and nucleus (17). Only in cells lacking both cytosolic TRXs was E GSH slightly more reduced in the nucleus compared to the cytosol.…”

The yeast oligopeptide transporter Opt2 is localized to peroxisomes and affects glutathione redox homeostasis

“…In the yeast Saccharomyces cerevisiae such probes have been used to measure glutathione redox changes in the cytosol (Braun et al, 2010), nucleus (Dardalhon et al, 2012), peroxisomes (Ayer et al, 2012) and mitochondria (Kojer et al, 2012). However not all subcellular compartments have been investigated and much remains to be understood regarding the partitioning of cellular glutathione homeostasis.…”