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Proposal

The purpose of this study was to understand the role of methylation in regulating the cellular stress response of Hsp31 in Saccharomyces cerevisiae yeast cells. Hsp31 is known to be methylated by the N-terminal methyltransferase Tae1. Changing the methylation site can affect the methylation status of Hsp31, which may play a role in the protective activity of Hsp31 against cellular stress. GLO1 is a gene in yeast involved in catalyzing the detoxification of methylglyoxal (MGO), which is a by-product of glycolysis. We established that S. cerevisiae in the glo1Δ and background is sensitive to cellular stress by MGO. Mutant strains in the glo1Δ background will simulate methylation levels, which can be used to determine if methylation increases or decreases the protective activity of Hsp31 under cellular stress. Hsp31 overexpression successfully rescues mutants in the glo1Δ background when treated with MGO. In a high throughput screen using CRISPR-based technology from Inscripta (Boulder, Colorado), a biotechnology company, we aim to replicate the results we observed in the glo1Δ background and potentially uncover new phenotypes in a series of experiments that will investigate the role of methylation in cellular processes in addition to oxidative stress, including heat stress and protein synthesis. The Inscripta library will allow us to collect data from approximately 1,000 mutants simultaneously rather than completing the experiment for each mutant individually. These results will provide much greater insight as to how methylation globally affects the cellular processes involved in the protective activity of Hsp31 and other substrates in S. cerevisiae.

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