Date of Award

Summer 2021

Document Type

Open Access Dissertation

Department

Chemistry and Biochemistry

First Advisor

Caryn E. Outten

Abstract

Thiol-disulfide redox homeostasis is integral for maintaining the redox status of proteins and other thiol-containing molecules within the cell. Among the many antioxidants and detoxifying enzymes and small peptides, glutathione (GSH) has proven to be critical for the preservation of function and structural integrity of the cell due to its functionality in areas like oxidative protein folding, thiol redox control, reactive oxygen species (ROS) and xenobiotics removal as well as iron regulation and Fe-S cluster biogenesis. Therefore, our studies are aimed at elucidating the factors that control GSH trafficking and metabolism in the model eukaryote Saccharomyces cerevisiae (baker’s yeast). Using genetic modifications combined with targeted redox-sensitive and pHsensitive green fluorescent protein probes, we have monitored GSH mediated pH changes and subsequently, changes in the redox potential, in the cytosol and mitochondrial matrix in response to fluctuating GSH levels in live yeast cells. These studies reveal that increased uptake of GSH or GSSG via overexpression of HGT1, which encodes a high affinity glutathione transporter, leads to significant changes in the intracellular pH in the cytosol and mitochondrial matrix, which, in turn, strongly impacts the redox state of GSH:GSSG pools. High intracellular GSH is known to be toxic for the cells, but it has not been shown how cells depleted of GSH (gsh1Δ) respond to increased GSH uptake. By expressing HGT1 in GSH-depleted gsh1Δ cells, we show that HGT1 overexpression partially rescues the inviability of GSH-deficient cells. Analysis of iron regulation and Fe-S cluster enzyme activity demonstrates that HGT1 overexpression reverses some of the Fe-S cluster biogenesis and iron homeostasis related defects in gsh1Δ strains. We also demonstrate that cysteine is a key amino acid for this rescue, suggesting that cysteine may partially substitute for GSH in gsh1Δ cells.

Included in

Chemistry Commons

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