Date of Award
Open Access Dissertation
Chemistry and Biochemistry
College of Arts and Sciences
Caryn E. Outten
The mitochondrial intermembrane space (IMS) is a unique subcellular compartment that houses key thiol-dependent redox pathways such as protein transport, mitochondrial respiration, and detoxification of ROS (reactive oxygen species). These pathways are all dependent on cysteine-rich proteins, thus maintaining thiol-disulfide balance in this organelle is crucial for cellular functions. An IMS protein import pathway called the Mia40-Erv1 disulfide relay system uses disulfide bond formation for the import and retention of substrate proteins in the IMS. Erv1 is also suggested to be involved in maturation of cytosolic Fe-S cluster proteins and regulation of iron homeostasis in S. cerevisiae. However, these studies were performed on one particular erv1 mutant strain (named as erv1-1) that we discovered has additional defects in glutathione (GSH) metabolism due to a secondary mutation in the gene encoding the GSH biosynthesis enzyme, Gsh1. Since the tripeptide GSH is also required for iron homeostasis and cytosolic Fe-S protein biogenesis, the Erv1-dependent connection between mitochondrial protein import, GSH metabolism, and iron homeostasis was investigated in several erv1 mutants. The GSH depletion phenotype was only detected in the erv1-1 strain and could be rescued by expressing GSH1 or adding GSH to the growth media. Additionally, expression of the iron uptake gene, FET3 and enzyme activities of Fe-S cluster proteins in several erv1 mutants were tested. Only the erv1-1 mutant has an iron misregulation defect, which could be rescued with GSH addition, and no significant effects on Fe-S cluster protein activities were detected. Our data suggests that the defects of cytosolic Fe-S maturation and iron regulation first reported in the erv1-1 strain is a direct consequence of GSH depletion rather than indicating a direct role for Erv1 in iron metabolism and cytosolic Fe-S cluster biogenesis.
We also characterized how mutations of Mia40 influence GSH metabolism and GSH:GSSG pools in the cytosol, mitochondrial matrix and intermembrane space. We have found that defects in Mia40 only influence the IMS redox state and do not alter cellular GSH levels. Additionally, we determined that defects in Mia40 do not impact iron homeostasis or Fe-S cluster biogenesis.
Furthermore, utilizing the roGFP2 in vivo sensors, we demonstrated how the deletion of manganese cofactor of superoxide dismutase 2 transporter Mtm1 and the citrate-oxoglutarate carrier Yhm2 affect the redox status of the mitochondrial matrix and IMS and cellular GSH levels. Deletion of MTM1 only leads to a large oxidative shift in the IMS GSH:GSSG redox state. In the contrary, deletion of YHM2 shows a smaller effect on the mitochondrial GSH:GSSG redox state even though, both mtm1Δ and yhm2Δ mutants were shown to have reduced mitochondrial GSH levels. Overall, we successfully characterized the roles of Erv1 and Mia40 in GSH metabolism, mitochondrial import and subcellular redox state which hereby helps to reveal their roles in Fe-S cluster biogenesis and iron regulation.
Ozer, H. K.(2015). The Role of Intermembrane Space Redox Factors In Glutathione Metabolism And Intracellular Redox Equilibrium. (Doctoral dissertation). Retrieved from https://scholarcommons.sc.edu/etd/3702