| Intracellular thiol-disulfide balance is very important for the activity of numerous enzymes and proteins that recruit cysteine residues as "thiol-switches". However, the redox balance can be disrupted by unregulated production of reactive oxygen species (ROS). Cells contain two primary redox regulatory systems that utilize thiol-disulfide redox chemistry to combat and detoxify ROS and help maintain thiol-disulfide balance: the glutathione (GSH)/glutathione disulfide (GSSG) redox couple and the reduced/oxidized thioredoxin (TRX) redox couple. Since glutathione has a relatively low redox potential and a high intracellular concentration (mM) relative to the other redox couples, it is considered to be the major thiol-disulfide redox buffer of the cell. The mitochondrion is an important compartmentalized organelle harboring numerous functions, while it is also the primary source and target of ROS. Numerous studies have shown that disruption of mitochondrial thiol redox circuits is related to cancer, neurodegenerative diseases, and aging. Therefore, knowledge of the fundamental mechanisms for maintaining thiol redox homeostasis in the mitochondria, especially a better understanding of mitochondrial GSH metabolism would aid in the development of therapies and novel drugs for treating mitochondrial diseases. However, the mechanisms are still poorly understood so far, and even less has been studied to compare the thiol redox states of the two mitochondrial sub-compartments. Previous researchers have developed a redox sensitive yellow fluorescence protein (rxYFP) which can specifically measure the in vivo redox state of the cytosolic GSH:GSSG. In this study we have modified rxYFP for expression in the mitochondrial intermembrane space (IMS) and matrix of the yeast Saccharomyces cerevisiae in order to compare thiol redox differences between these compartments and the cytosol. It was demonstrated that IMS is considerably more oxidizing than the cytosol or matrix. These sensors' dynamic response to an exogenous oxidant and reductant were confirmed, and their responses to specific redox changes that are localized to subcellular compartments were verified by manipulating the subcellular redox status using GSH reductase mutants. These studies indicate that redox control is independently regulated within these individual compartments in the cell. Moreover, to further optimize and verify the usage of these redox sensors, the effects of different factors on the redox measurements were investigated and the methodology were optimized and summarized as well. These studies represent an important step towards understanding the mechanisms of mitochondrial thiol redox homeostasis. |
