Date of Award
Campus Access Thesis
Chemistry and Biochemistry
Caryn E Outten
Redox homeostasis is essential for all living organisms. Maintaining thiol-disulfide balance is especially important for the mitochondrion, since it is the main source and target of redox oxygen species (ROS) that can alter the balance. To gauge the redox states of the mitochondrial matrix and intermembrane space, we targeted green fluorescent protein (GFP) based redox and pH sensors to these specific compartments in the model organism Saccharomyces cerevisiae. The correct localization was verified by subcellular fractionation and western blot. These genetically engineered sensors allow us to determine the in vivo pH and redox change of distinct subcellular compartments.
By using these sensors, we studied the pH change upon adding glutathione (GSH) to HGT1 (High-affinity glutathione transporter 1) transformed yeast cells. We successfully determined the pH change with high accuracy and sensitivity upon adding GSH/GSSG to the media. The results demonstrate that HGT1 co-transports GSH and H+. To test the effect of endogenous GSH:GSSG upon the change of pH, we also used a glr1Ä strain (GLR1 is a gene encoding both cytosolic and mitochondrial forms of glutathione reductase) and found no significant difference in pH between the wild type cells and glr1Ä cells.
Glutathione and mitochondrion also play a significant role in the maintenance of iron homeostasis in yeast. Since the mitochondrion is the sole site of heme synthesis and the majority of iron sulfur cluster (ISC) generation, it is a focal point of iron metabolism. Previous research has indicated that the mitochondrial ISC assembly machinery is required for the maturation of both mitochondrial and cytosolic Fe-S proteins. The IMS-localized sulfhydryl oxidase Erv1 and GSH are both proposed to be involved in cytosolic iron-sulfur protein biogenesis. To reveal the mechanism of this connection, we measured the total GSH in several temperature-sensitive erv1/mia40 mutants. Mia40 is an import receptor that functions with Erv1 to oxidize cysteines in the IMS. Our results show that cytosolic GSH depletion is found only in one particular erv1 mutant and none of the mia40 mutants. Thus, the Mia40-Erv1 IMS import path way may not be directly involved in cytosolic Fe-S protein maturation as proposed before. Instead, specific mutations in erv1 may influence GSH levels, which in turn cause disruption of cytosolic Fe-S cluster maturation.
XUE, Z.(2011). Using GFP-Based Redox and pH Sensors to Monitor Glutathione Metabolism In the Mitochondrial Intermembrane Space. (Master's thesis). Retrieved from http://scholarcommons.sc.edu/etd/747