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Abstract
Many environmental stressors disrupt protein homeostasis in the eukaryotic cell. To adapt, the eukaryotic cell activates the heat shock response which, in S.cerevisiae, involves the induction of 42 target genes, many of which are protein-folding chaperones. The induced target genes, collectively called the heat shock response (HSR), replenish the cell’s supply of protein folding machinery, so that there are sufficient chaperones to fold the proteins in the cell. HSR hyperactivity and hypoactivity in human disease states indicates that HSR regulation in both directions is essential for normal cell functioning, motivating this project’s primary question: how does a healthy cell respond and adapt to unexpected changes in conditions? What feedback mechanisms tune the HSR level?
In the pilot study presented in Chapter 2, one Hsf1 target, Sis1, is investigated for a potential role in feedback regulation of the HSR. As Sis is necessary to repress Hsf1 during non stress, its induction during stress might be necessary for Hsf1 deactivation. Surprisingly, Sis1 induction is dispensable for Hsf1 deactivation, but instead supports long term fitness. Through this initial study, we developed a long-term quantitative fitness assay, and we came to appreciate that the HSR not only restores homeostasis, but supports cell adaptation and growth resumption.
In Chapter 3, all HSR target genes were screened for their role in feedback regulation. Induction of six Hsf1 target genes impacts Hsf1 activity, though none as dramatically as the Hsf1 target Hsp70, identified previously. Induction of four of the six Hsf1 target genes was important for clearing cytosolic protein aggregates during heat shock, indicating these genes regulate Hsf1 activity by boosting Hsp70 availability. Follow up investigations revealed that the most significant novel feedback regulator, Fes1, is indeed necessary for relocalization of Hsp70 from protein aggregates in the cytosol to the nucleus. In conclusion, all feedback regulation on Hsf1 seems to proceed via the Hsp70-dependent negative feedback loop.
Chapter 4 is an investigation of Sis1’s role at orphan ribosomal protein (oRP) aggregates at the nucleolar periphery. First author Asif Ali found that Sis1 maintains the dynamic, liquid-like state of condensates, and is necessary for efficient condensate dispersal after stress. To investigate whether the Sis1-dependent reversibility of condensates had physiological relevance, Sis1 is transiently depleted from the nucleus during heat shock, then cell fitness is measured. Transient Sis1 depletion during heat shock affects cells’ ability to resume growth quickly after heat shock so Sis1-maintained oRP condensates are necessary for efficient growth resumption after stress. This study begins to elucidate endogenous, stress-induced protein aggregates: their contents, function, and the chaperones which mediate their formation and resolution.
We now know that heat shock response feedback regulation architecture is simple, and centered around the Hsp70-dependent negative feedback loop. Its simplicity probably facilitates the conservation of this regulatory mechanism across eukaryotes, which likely receives input from diverse proteins across the cell, in response to diverse stressors.