Investigating Subcellular Thiol Redox Chemistry With Gfp-Based Redox Sensors

The mitochondrion is an essential cellular organelle that houses critical metabolic pathways such as respiration, Fe-S cluster and heme biosynthesis, and biosynthesis of lipids, amino acids and nucleotides. These pathways are all dependent on cysteine-containing proteins, thus maintaining thiol-disulfide balance in this organelle is critical for cellular function. Thiol-disulfide equilibrium is primarily controlled by the reduced and oxidized forms of the abundant tripeptide glutathione (GSH and GSSG), which serve as an intracellular redox buffer. In order to better understand the factors that influence mitochondrial GSH:GSSG balance, we used genetic engineering methods to target fluorescent protein-based redox sensors to the matrix and intermembrane space (IMS) of yeast mitochondria. This approach allows us to separately monitor the in vivo redox state of the matrix and IMS, providing a more detailed picture of redox processes in these two compartments. The two sensors employed (rxYFP, or redox-sensitive yellow fluorescent protein and roGFP, or redox-sensitive green fluorescent protein) specifically equilibrate with the local GSH:GSSG pool and register redox changes via disulfide bond formation. Redox western and fluorimeter measurements were used to demonstrate that the GSH:GSSG redox status of the mitochondrial IMS is maintained separately from the cytosol and matrix. Furthermore, our lab was the first to demonstrate that this subcellular compartment is more oxidizing than the cytosol and mitochondrial matrix. Glutathione reductase (Glr1) is responsible for the regeneration of GSH to maintain the reduced environment. Experiments show that expressing IMS-localized Glr1 does not reduce the IMS. This data suggests that IMS redox state is oxidized not because of lacking Glr1 but by some other unknown mechanism. Using the rxYFP in vivo sensors, we have also found that the mutations in an IMS-localized sulfhydryl oxidase (Erv1) leads to reduced levels of GSH in the cytosol and matrix, demonstrating that IMS protein function can have direct consequences on the overall cellular redox state. The sensors were also used to monitor temporal and spatial changes in GSH:GSSG equilibrium upon GSH or GSSG overaccumulation. Overall, these tools allow for non-destructive, real-time redox potential measurements in subcellular compartments and may help to elucidate the mechanisms for maintaining mitochondrial redox balance