Oxidative stress mechanism in yeast presents an innovative pathway to understand in creating the next generation of antifungal drugs. Rgc1 and Rgc2 are paralogous proteins that regulate the Fps1 glycerol channel in hyperosmotic stress. Hyperosmotic conditions lead Hog1 MAP kinase to phosphorylate Rgc2 and cause its dissociation from Fps1, allowing the channel to close and protect the cell from damage. Rgc2 contains pleckstrin homology (PH) domains broken up by long insertions and more phosphorylation sites than targeted by Hog1 in response to hyperosmotic stress. Since none of the other MAP kinases in yeast were seen to phosphorylate Rgc2 during oxidative stress, it is thought that Rgc2 may bind to other proteins. In this study, the sensitivity of a strain deleted for both RGC1 and RGC2 was compared to strains with single deletions in either gene in response to oxidative stress. Having deletions in both RGC1 and RGC2 caused increased sensitivity to hydrogen peroxide whereas strains with deletions in either gene seemed unaffected, correlating with the fact that Rgc1 and Rgc2 are paralogous proteins, able to recover each other's functions. A second analysis compared mutated Fps1 (fps1∆-FKSV) and a strain with deletions for both RGC1 and RGC2 (rgc1/2∆). The fps1∆-FKSV strain has four amino acid substitutions in the C-terminal region where Rgc2 binds to Fps1. While both strains grew less than wild-type in hydrogen peroxide, the rgc1/2∆ strain was more sensitive suggesting that Rgc1/2 has an additional role in oxidative stress. To identify the oxidative stress function of Rgc1/2, a genomic overexpression library was transformed into the rgc1/2∆ strain and used for a suppressor screen in the presence of hydrogen peroxide. Although the screen revealed a manageable amount of 49 candidates, only four produced sequences that spanned a protein-encoding region. The candidate plasmids were transformed back into the rgc1/2∆ strain for preparation of a sensitivity assay which showed that the colonies did not survive any better than the starting rgc1/2∆ strain. Without a plausible plasmid candidate, we decided to look into the effect of YAP1 on the rgc1/2∆ strain. Yap1 is a transcription factor known to activate many genes in oxidative stress. Two forms of YAP1 were transformed into rgc1/2∆: wild-type YAP1 and YAP1-A627E which contains a mutation in the nuclear export signal. Compared to the controls, YAP1-A627E allowed the rgc1/2∆ strain to grow at 1.5mM H2O2 while wild-type YAP1 did not. This result showed that a constitutively nuclear Yap1 can overcome deletions in RGC1 and RGC2. It also suggested that an increased activity in the nucleus was important in hydrogen peroxide resistance and another suppressor screen of rgc1/2∆ was performed looking for spontaneous mutations in the genomic DNA. The screened colonies were tested for their survival on hydrogen peroxide but their resistance appeared to be transient. We have shown Rgc1 and Rgc2 to be important cellular components in oxidative stress in addition to hyperosmotic stress. Further research on Rgc1/2 would provide invaluable knowledge on oxidative stress protection in yeast and a better foundation on which to build antifungal drugs.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/15092 |
| Date | 22 January 2016 |
| Creators | Tsai, Michelle |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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