Regulation of Endoplasmic Reticulum Stress in Saccharomyces cerevisiae
Abstract
The budding yeast Saccharomyces cerevisiae has been used extensively to uncover the genetic mechanisms that control basic cellular processes, including survival, maintenance, and response to stressors. One metric of yeast survival is chronological lifespan (CLS), which is the amount of time non-dividing yeast cells can survive at stationary phase. Variations in CLS following genetic alteration are used to understand the function of specific genes and pathways in cellular aging. Many factors contribute to aging, including accumulation of toxic misfolded secretory proteins in the endoplasmic reticulum (ER stress), to which the cell responds through activation of ER stress signaling pathways, such as the Unfolded Protein Response (UPR).
In this thesis, we first developed new fluorescent assays and a corresponding software program to measure yeast CLS and investigate how ER stress responses impact yeast CLS. Using these assays, we found that inositol was important for CLS, especially in cells with compromised UPR. We also found that UPR-dependent upregulation of the ER chaperone Kar2 is critical for CLS, and we demonstrate that deletion of ER-associated degradation (ERAD) components accelerates chronological aging. We argue that the capacity of the ER quality control machinery to degrade misfolded secretory proteins is an important determinant of ER stress sensitivity and, by extension, yeast CLS. Finally, we aimed to identify ways to modulate ER stress and the UPR; we worked to decipher the mechanism of a compound previously hypothesized to act as a “chemical chaperone” to alleviate ER stress by directly improving protein folding, trafficking, and degradation – tauroursodeoxycholic acid, or TUDCA. We found that, while TUDCA alleviates ER stress, this process can be uncoupled from UPR signaling. We also found little evidence that TUDCA works as a true chemical chaperone. By contrast, it alleviates ER stress indirectly through activation of another stress response, the Cell Wall Integrity (CWI) pathway. We thus discovered a novel mechanism of modulating ER stress. Overall, this thesis has identified new factors influencing ER stress regulation, including chronological aging, protein quality control mechanisms, the CWI pathway, and small molecules such as TUDCA.