Date of Graduation

5-2021

Document Type

Thesis

Degree Name

Bachelor of Science

Degree Level

Undergraduate

Department

Biological Sciences

Advisor/Mentor

Lewis, Jeffrey

Committee Member/Reader

Sakon, Josh

Committee Member/Second Reader

Evans, Timothy

Committee Member/Third Reader

Fan, Chenguang

Abstract

Evidence is emerging that protein lysine acetylation may be a novel type of post-translational modification (PTM) contributing to the mechanisms of yeast heat stress responses. Proteomics studies including ours have identified over 1,000 acetylated proteins in the yeast proteomes that are composed of about 6,000 proteins. Our lab recently identified 596 proteins that underwent acetylation changes during heat shock by mass spectrometry. However, the role of lysine acetylation on specific residues of specific proteins in yeast thermotolerance remains largely unknown. This study selected 43 proteins from our lab’s previous work and examined their possible contributions to yeast heat stress responses. We found that knockout of 32 genes caused a growth defect in yeasts at 40 °C, suggesting these proteins are required for yeast innate thermotolerance. Among these 32 proteins, knockout of 5 genes including rpl31a, sin3, aco1, adh1 and pfk2 almost completed inhibited yeast growth at 40 °C, suggesting they are ideal candidates for further studies. Site-directed mutagenesis method was employed to replace the lysine K638 in Aco1p protein for the purpose of mimicking different states of acetylation; K638 was first replaced with alanine to examine whether this lysine residue was essential to yeast survival and growth, and then K638 was replaced by glutamine or arginine to mimic acetylated or un-acetylatable Aco1p, respectively. Similar work was performed for proteins Rpl31ap, Sin3p, and Hsp104p, whose function was confirmed in this study to be required for yeast innate and acquired thermotolerance. Work is in progress to examine if these manipulations will impact yeast thermotolerance. To conclude, this study identified that 32 protein with changing acetylation are required for yeast innate thermotolerance. In addition, this work generated mutant strains 4 harboring desired residues that are useful to examine the role of specific lysine residues whose acetylation may play a role in regulating the yeast heat stress responses.

Keywords

s. cerevisiae; heat shock; gene knockout; site-directed mutagenesis

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