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.

Peroxiredoxin (Prx) 1 is a member of the thiol-specific peroxidases family and plays diverse roles such as H2O2 scavenger, redox signal transducer and molecular chaperone. Prx1 has been reported to be involved in protecting cancer cells against various therapeutic challenges. We investigated how modulations of intracellular redox system affect cancer cell sensitivity to reactive oxygen species (ROS)-generating drugs. We observed that stable and transient Prx1 knockdown significantly enhanced HeLa cell sensitivity to β-lapachone (β-lap), a potential anticancer agent. Prx1 knockdown markedly potentiated 2 µM β-lap-induced cytotoxicity through ROS accumulation. This effect was largely NAD(P)H:quinone oxidoreductase 1 dependent and associated with a decrease in poly(ADP-ribose) polymerase 1 protein levels, phosphorylation of JNK, p38 and Erk proteins in mitogen-activated protein kinase (MAPK) pathways and a decrease in thioredoxin 1 (Trx1) protein levels. Trx1 serves as an electron donor for Prx1 and is overexpressed in Prx1 knockdown cells. Based on the fact that Prx1 is a major ROS scavenger and a partner of at least ASK1 and JNK, two key components of MAPK pathways, we propose that Prx1 knockdown-induced sensitization to β-lap is achieved through combined action of accumulation of ROS and enhancement of MAPK pathway activation, leading to cell apoptosis. These data support the view that modulation of intracellular redox state could be an alternative approach to enhance cancer cell sensitivity to ROS-generating drugs or to overcome some types of drug resistance.