Nuclear and cytosolic J-domain proteins Apj1, Sis1, and Ydj1 provide synergistic control of Hsf1 at distinct phases of the heat shock response

The cellular protein quality control (PQC) system prevents the buildup of misfolded proteins by refolding, degrading, and sequestering them, thereby maintaining protein homeostasis, also known as proteostasis. The heat shock response (HSR) is a transcriptional mechanism that controls the expression of the stress response machinery, which protects eukaryotic cells from proteotoxic stress in both the cytosol and nucleus. The initiation and regulation of the response are mediated by stress-dependent control of heat shock transcription factors (HSFs). Saccharomyces cerevisiae encodes a single HSF (Hsf1), making it easier to study HSR regulation. Hsf1 is suppressed by Hsp70 chaperones under normal, nonstress conditions and becomes activated during proteotoxic stress, directly linking protein misfolding and its recovery to the HSR. J-domain proteins (JDPs) are crucial for targeting of Hsp70s to their substrates, yet the specific JDP(s) responsible for regulating Hsf1 and connecting protein damage to HSR activation and inactivation (attenuation) were unclear. In yeast, 22 JDP proteins exist, 11 of which are located in the cytosol and nucleus. They include the nuclear Apj1 and cytonuclear Ydj1 and Sis1. The class A JDP Apj1 is expressed in low copy numbers under non-stress conditions and disaggregates substrates within the intra-nuclear protein quality control compartment (INQ) during proteotoxic stress. The nuclear and cytosolic class B JDP Sis1 has been suggested as the main regulator of HSR induction, yet it is not implicated in HSR attenuation. Class A Ydj1 is the most abundant JDP in the yeast cytosol, and its deletion results in a strong proteostasis phenotype without inducing the HSR. In my doctoral thesis, I show that deletion of the gene encoding the yeast nuclear JDP Apj1 results in significantly impaired HSR attenuation. In apj1Δ cells, this leads to upregulation of heat shock protein expression, which is primarily controlled by Hsf1, after 60 minutes of continuous heat stress. Additionally, I see colocalization of Hsf1-GFP and Apj1-mScarlet during the HSR. ChIP-Seq data from our collaboration partners Dr. Fabian den Brave (University of Bonn) and Prof. David Gross (LSU Shreveport) showed that Apj1 interacts with DNA-bound Hsf1 during the attenuation of the HSR. The combined data show that Apj1 controls the attenuation phase of the HSR by promoting Hsf1’s displacement from heat shock elements in promoter regions. Apj1’s involvement in both nuclear PQC and Hsf1 regulation suggests a direct link between nuclear proteostasis and HSR regulation. Sis1 has been implicated in repressing V Hsf1 prior to stress. However, Sis1 depletion led to enhanced expression of only some Hsf1-controlled genes, arguing against a dominant role in repressing Hsf1 in non-stressed cells. Additionally, combined depletion of both Sis1 and Apj1 did not result in a greater impact on HSR induction or attenuation, suggesting that the two JDPs function at different stages of the HSR. The lack of a synergistic effect from the APJ1 SIS1 double mutant suggests that other JDPs are involved in Hsf1 regulation under non-stress conditions. In fact, depleting Ydj1 and Apj1 simultaneously induces a strong HSR, indicating that Ydj1 plays a key role as a JDP that suppresses Hsf1 in non-stressed cells. I tested the hypothesis that the strong HSR phenotype of Ydj1-Apj1-deficient mutant cells is due to Ydj1's major role in protein homeostasis, leading to Sis1 titration. Cells lacking Ydj1 experience a proteostasis defect, resulting in a temperature-sensitive growth phenotype, but surprisingly, the HSR is not activated in these cells. Depleting Apj1 alongside Ydj1 restores proteostasis and alleviates growth sensitivity in YDJ1 mutant cells by inducing HSR upregulation. This indicates that Apj1 prevents the adjustment of chaperone levels to compensate for proteostasis defects by repressing Hsf1 in the YDJ1 mutant strain. Transcription and translation of genes controlled by Hsf1 in the nonstressed APJ1 YDJ1 double mutant are similar to those during heat shock. At the same time, ribosome biogenesis is reduced in the APJ1 YDJ1 double mutant, pointing to an intertwined activation of heat shock genes and suppression of ribosome production. Misfolded proteins in the cytosol can be directed to the nucleus for degradation. I propose that Ydj1 regulates the HSR by transporting misfolded proteins into the nucleus, where Apj1 can detect them, thereby connecting cytosolic to nuclear proteostasis and HSR control. To summarize, I show that in yeast S. cerevisiae, three JDPs—Apj1, Sis1, and Ydj1— work together to regulate the Hsf1-dependent HSR. Sis1 and Ydj1 maintain proteostasis under non-stress conditions, while all three JDPs suppress Hsf1. A decline in proteostasis activates the HSR by titrating away JDPs. Ydj1 could link the proteostasis state of the nucleus to that of the cytosol, while Apj1 connects the shutdown of the HSR to the proteostasis state within the nucleus.