Download the role of intermembrane space redox factors in glutathione

Hatice Kubra Ozer
Chemistry and Biochemistry, PhD
THE ROLE OF INTERMEMBRANE SPACE REDOX FACTORS IN
GLUTATHIONE METABOLISM AND INTRACELLULAR REDOX
EQUILIBRIUM
ABSTRACT
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
mtm1∆ and yhm2∆ mutants was 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.