Date of Award
Open Access Dissertation
Chemistry and Biochemistry
Caryn E. Outten
Thomas M. Makris
Thiol-disulfide redox homeostasis is integral for maintaining the redox status of proteins and other thiol-containing molecules within cells. Mitochondria are particularly vulnerable to disruptions in redox balance, due to production of reactive oxygen species (ROS) during oxidative metabolism. Among the many antioxidants and detoxifying enzymes existing in mitochondria, glutathione (GSH) has proven to be critical for the preservation of function and structural integrity of the organelle. Therefore, our studies are aimed at elucidating the factors that control mitochondrial GSH metabolism in the model eukaryote Saccharomyces cerevisiae (baker’s yeast). A critical component of understanding subcellular GSH homeostasis is identifying routes of entry into the organelle. Using genetic modifications combined with targeted redox-sensitive green fluorescent protein-based probes, previous studies found that GSH enters the mitochondrial intermembrane space (IMS) through porins located in the outer mitochondria membrane via passive diffusion. These studies demonstrated that reduced (GSH) and oxidized glutathione (GSSG) pools of the cytosol and IMS are interconnected. On the contrary, the GSH redox potential (EGSH) of the mitochondrial matrix was found to be separate from that of the cytosol. While attempts have been made to uncover a mitochondrial GSH/GSSG importer, none have been confirmed with certainty. Our current studies are aimed at identifying a mitochondrial transporter specific for GSH and biological processes that affect mitochondrial GSH/GSSG pools. Furthermore, we tested the effects of GSH and GSSG overaccumulation on the mitochondria of yeast cells overexpressing the high affinity GSH/GSSG transporter, Hgt1. We also explored the possibility that plasma membrane-localized Hgt1 exhibits dual localization to the mitochondrial membrane. We find that HGT1 overexpression in strains incubated with GSH and GSSG leads to GSH and GSSG accumulation in mitochondria as well as the cytosol. However, Hgt1 does do not appear to be localized to mitochondria. Intercompartmental cross-talk is also thought to regulate subcellular GSH:GSSG pools. Therefore, we investigated if vacuolar storage of excess GSH could affect mitochondrial GSH concentrations. Our subcellular GSH measurements imply that HGT1 overexpression affects mitochondrial GSH pools and the vacuole acts as a buffering compartment during GSH and GSSG overaccumulation.
The physiological role of GSH depletion on cellular function and subcellular redox status has been well characterized. Mitochondrial GSH has been reported to be critically important because of its roles in redox homeostasis and iron metabolism. High intracellular GSH has also been shown to be toxic to cells, yet it has not been determined how cells depleted of GSH (gsh1∆) respond to increased GSH uptake. To study this, we employed a gsh1∆ yeast strain engineered to overexpress HGT1. Interestingly, our results show that HGT1 overexpression alone can partially rescue growth in cells devoid of GSH and reverse some of the iron-related phenotypes. We demonstrate that cysteine is a key amino acid for rescue, suggesting that cysteine may partially substitute for GSH in gsh1∆ cells. However, we did not find significantly increased cysteine levels in HGT1 overexpressing strains, leaving open the specific mechanism whereby HGT1 overexpression compensates for the lack of GSH.
McGee, C. C.(2019). Impact of Glutathione Transporters on Subcellular Glutathione Pools and Cell Survival in Saccharomyces Cerevisiae. (Doctoral dissertation). Retrieved from https://scholarcommons.sc.edu/etd/5133