Heat Shock Factor 1 (Hsf1) in yeast drives the basal transcription of key proteostasis factors and its activity is induced as part of the core heat shock response. Exploring Hsf1 specific functions has been challenging due to the essential nature of the HSF1 gene and the extensive overlap of target promoters with environmental stress response (ESR) transcription factors Msn2 and Msn4 (Msn2/4). In this study, we constructed a viable hsf1 increment strain by replacing the HSF1 open reading frame with genes that constitutively express Hsp40, Hsp70, and Hsp90 from Hsf1-independent promoters. Phenotypic analysis showed that the hsf1 increment strain grows slowly, is sensitive to heat as well as protein misfolding and accumulates protein aggregates. Transcriptome analysis revealed that the transcriptional response to protein misfolding induced by azetidine-2-carboxylic acid is fully dependent on Hsf1. In contrast, the hsf1 increment strain responded to heat shock through the ESR. Following HS, Hsf1 and Msn2/4 showed functional compensatory induction with stronger activation of the remaining stress pathway when the other branch was inactivated. Thus, we provide a long-overdue genetic test of the function of Hsf1 in yeast using the novel hsf1 increment construct. Our data highlight that the accumulation of misfolded proteins is uniquely sensed by Hsf1-Hsp70 chaperone titration inducing a highly selective transcriptional stress response.

The mitochondrial genome is responsible for the production of a handful of polypeptides that are core subunits of the membrane-bound oxidative phosphorylation system. Until now the mechanistic studies of mitochondrial protein synthesis inside cells have been conducted with inhibition of cytoplasmic protein synthesis to reduce the background of nuclear gene expression with the undesired consequence of major disturbances of cellular signaling cascades. Here we have generated a system that allows direct monitoring of mitochondrial translation in unperturbed cells. A recoded gene for superfolder GFP was inserted into the yeast (Saccharomyces cerevisiae) mitochondrial genome and enabled the detection of translation through fluorescence microscopy and flow cytometry in functional mitochondria. This novel tool allows the investigation of the function and regulation of mitochondrial translation during stress signaling, aging and mitochondrial biogenesis.

Cellular proteostasis ismaintained via the coordinated synthesis, maintenance, and breakdown of proteins in the cytosol and organelles. While biogenesis of the mitochondrial membrane complexes that execute oxidative phosphorylation depends on cytoplasmic translation, it is unknown how translation within mitochondria impacts cytoplasmic proteostasis and nuclear gene expression. Here we have analyzed the effects of mutations in the highly conserved accuracy center of the yeast mitoribosome. Decreased accuracy of mitochondrial translation shortened chronological lifespan, impaired management of cytosolic protein aggregates, and elicited a general transcriptional stress response. In striking contrast, increased accuracy extended lifespan, improved cytosolic aggregate clearance, and suppressed a normally stress-induced, Msn2/4-dependent interor-ganellar proteostasis transcription program (IPTP) that regulates genes important for mitochondrial proteostasis. Collectively, the data demonstrate that cytosolic protein homeostasis and nuclear stress signaling are controlled by mitochondrial translation efficiency in an inter-connected organelle quality control network that determines cellular lifespan.

Protein quality control depends on the tight regulation of interactions between molecular chaperones and polypeptide substrates. Substrate release from the chaperone Hsp70 is triggered by nucleotide-exchange factors (NEFs) that control folding and degradation fates via poorly understood mechanisms. We found that the armadillo-type NEFs budding yeast Fes1 and its human homolog HspBP1 employ flexible N-terminal release domains (RDs) with substrate-mimicking properties to ensure the efficient release of persistent substrates from Hsp70. The RD contacts the substrate-binding domain of the chaperone, competes with peptide substrate for binding and is essential for proper function in yeast and mammalian cells. Thus, the armadillo domain engages Hsp70 to trigger nucleotide exchange, whereas the RD safeguards the release of substrates. Our findings provide fundamental mechanistic insight into the functional specialization of Hsp70 NEFs and have implications for the understanding of proteostasis-related disorders, including Marinesco-Sjögren syndrome.

Protein aggregation is intimately associated with cellular stress and is accelerated during aging, disease, and cellular dysfunction. Yeast cells rely on the ATP-consuming chaperone Hsp104 to disaggregate proteins together with Hsp70. Hsp110s are ancient and abundant chaperones that form complexes with Hsp70. Here we provide in vivo data showing that the Saccharomyces cerevisiae Hsp110s Sse1 and Sse2 are essential for Hsp104-dependent protein disaggregation. Following heat shock, complexes of Hsp110 and Hsp70 are recruited to protein aggregates and function together with Hsp104 in the disaggregation process. In the absence of Hsp110, targeting of Hsp70 and Hsp104 to the aggregates is impaired, and the residual Hsp104 that still reaches the aggregates fails to disaggregate. Thus, coordinated activities of both Hsp104 and Hsp110 are required to reactivate aggregated proteins. These findings have important implications for the understanding of how eukaryotic cells manage misfolded and amyloid proteins.

Proteins have to be folded to their native structures to be functionally expressed. Misfolded proteins are proteotoxic and negatively impact on cellular fitness. To maintain the proteome functional proteins are under the constant surveillance of dedicated molecular chaperones that perform protein quality control (PQC). Using the model organism yeast Saccharomyces cerevisiae this thesis investigates the molecular mechanisms that cells employ to maintain protein homeostasis (proteostasis). In Study I the role of the molecular chaperone Hsp110 in the disentanglement and reactivation of aggregated proteins was investigated. We found that Hsp110 is essential for cellular protein disaggregation driven by the molecular chaperones Hsp40, Hsp70 and Hsp104 and characterized its involvement via regulation of Hsp70 ATPase activity as a nucleotide exchange factor. In Study II we found out that Hsp110 undergoes translational frameshifting during its expression resulting in a nuclear targeting. Nuclear Hsp110 interacts with Hsp70 and reprograms the proteostasis system to better deal with stress and to confer longevity. Study III describes regulation of Hsp70 function in PQC by the nucleotide exchange factor Fes1. We found that rare alternative splicing regulates Fes1 subcellular localization in the cytosol and nucleus and that the cytosolic isoform has a key role in PQC. In Study IV we have revealed the molecular mechanism that Fes1 employ in PQC. We show that Fes1 carries a specialized release domain (RD) that ensures the efficient release of protein substrates from Hsp70, explaining how Fes1 maintains the Hsp70-chaperone system clear of persistent misfolded proteins. In Study V we report on the use of a novel bioluminescent reporter (Nanoluc) for use in yeast to measure the gene expression and protein levels. In summary, this thesis contributes to the molecular understanding of chaperone-dependent PQC mechanisms both at the level of individual components as well as how they interact to ensure proteostasis.

Cells maintain proteostasis by selectively recognizing and targeting misfolded proteins for degradation. In Saccharomyces cerevisiae, the Hsp70 nucleotide exchange factor Fes1 is essential for the degradation of chaperone-associated misfolded proteins by the ubiquitin-proteasome system. Here we show that the FES1 transcript undergoes unique 3' alternative splicing that results in two equally active isoforms with alternative C-termini, Fes1L and Fes1S. Fes1L is actively targeted to the nucleus and represents the first identified nuclear Hsp70 nucleotide exchange factor. In contrast, Fes1S localizes to the cytosol and is essential to maintain proteostasis. In the absence of Fes1S, the heat-shock response is constitutively induced at normally non-stressful conditions. Moreover, cells display severe growth defects when elevated temperatures, amino acid analogues or the ectopic expression of misfolded proteins, induce protein misfolding. Importantly, misfolded proteins are not targeted for degradation by the ubiquitin-proteasome system. These observations support the notion that cytosolic Fes1S maintains proteostasis by supporting the removal of toxic misfolded proteins by proteasomal degradation. This study provides key findings for the understanding of the organization of protein quality control mechanisms in the cytosol and nucleus.

Reporter proteins are essential tools in the study of biological processes and are employed to monitor changes in gene expression and protein levels. Luciferases are reporter proteins that enable rapid and highly sensitive detection with an outstanding dynamic range. Here we evaluated the usefulness of the 19 kDa luciferase NanoLuc (Nluc), derived from the deep sea shrimp Oplophorus gracilirostris, as a reporter protein in yeast. Cassettes with codon-optimized genes expressing yeast Nluc (yNluc) or its destabilized derivative yNlucPEST have been assembled in the context of the dominant drug resistance marker kanMX. The reporter proteins do not impair the growth of yeast cells and exhibit half-lives of 40 and 5 min, respectively. The commercial substrate Nano-Glo (R) is compatible with detection of yNluc bioluminescence in < 50 cells. Using the unstable yNlucPEST to report on the rapid and transient expression of a heat-shock promoter (PCYC1-HSE), we found a close match between the intensity of the bioluminescent signal and mRNA levels during both induction and decay. We demonstrated that the bioluminescence of yNluc fused to the C-terminus of a temperature-sensitive protein reports on its protein levels. In conclusion, yNluc and yNlucPEST are valuable new reporter proteins suitable for experiments with yeast using standard commercial substrate.