Date of Award

2016

Document Type

Open Access Thesis

Department

Chemistry and Biochemistry

Sub-Department

College of Arts and Sciences

First Advisor

Caryn E. Outten

Abstract

Iron homeostasis in Saccharomyces cerevisiae is controlled through several pathways and is important because too much iron can cause cellular damage. Iron homeostasis in yeast is primarily controlled by transcription factors Aft1 and Aft2 which bind to the iron regulon to induce iron uptake when iron levels are low. When iron levels are sufficient, Aft1 and Aft2 are bound and inhibited by monothiol glutaredoxins (Grxs) Grx3 and Grx4. These Grxs are also involved in iron trafficking in the cell and bind to and deliver iron-sulfur clusters to multiple proteins.

The first part of this thesis focuses on determining if excess GSH/GSSG disrupts iron trafficking in grx3 grx4 mutant strains (grx3Δgrx4Δ) and (grx3Δ Gal-GRX4). Total and oxidized glutathione were increased in Δgrx3Δgrx4 compared to wild type (WT). Total and oxidized glutathione was higher in Δgrx3 Gal-GRX4 than WT 40 hours after galactose removal, but by 64 hours a suppressor mutation developed and glutathione levels resembled those of WT. Δgrx4 Gal-GRX3 had the same issue. The increased glutathione previously observed in literature were not able to be replicated. Experiments of expression levels of GSH1 were inconclusive.

The second part of this thesis looks at iron homeostasis in budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe. These species utilize homologous proteins as well as proteins unique to each species. In S. pombe, glutaredoxin, Grx4, interacts with and regulates the iron-dependent transcriptional repressor Php4. Similar to its homologues, Grx4 forms a [2Fe-2S]-bridged homodimer alone, and a [2Fe-2S]-bridged heterocomplex when co-expressed with Php4. When iron is sufficient, Grx4 interacts with Php4 to form a [2Fe-2S] cluster-bound complex, communicating cellular iron status and inhibiting Php4 activity. Fra2 was often insoluble and unstable. Coexpressing Fra2 with Pphp4 or Grx4 did not improve the stability. A urea extraction of Fra2 expressed independently improved solubility but Fra2 was still unstable. Fra2 coexpressed with the Takara chaperones did not improve the solubility.

The third portion of this thesis involves glutathione reducing reactive oxygen species (ROS) and maintaining redox balance within the cell. A rxYFP sensor was targeted to the mitochondrial intermembrane space (IMS) in order to measure the redox status of this subcellular compartment when the cell was exposed to oxidative stress. The hope for this work is to answer questions about the redox states and mechanisms controlling redox homeostasis on the subcellular level. The rxYFP sensor in the HGT1 overexpression strain as well as in the empty vector control was much more reduced than previously observed. Troubleshooting experiments were unable to resolve this issue.

Rights

© 2016, Kirsten Renee Collins

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