Besides their famous role as powerhouse of the cell, mitochondria are also involved in many signaling processes and metabolism. Therefore, it is unsurprising that mitochondria are no isolated organelles but are in constant crosstalk with other parts of the cell. Due to the endosymbiotic origin of mitochondria, they still contain their own genome and gene expression machinery. The mitochondrial genome of yeast encodes eight proteins whereof seven are core subunits of the respiratory chain and ATP synthase. These subunits need to be assembled with subunits imported from the cytosol to ensure energy supply of the cell. Hence, coordination, timing and accuracy of mitochondrial gene expression is crucial for cellular energy production and homeostasis. Despite the central role of mitochondrial translation surprisingly little is known about the molecular mechanisms.

In this work, I used baker’s yeast Saccharomyces cerevisiae to study different aspects of mitochondrial translation. Exploiting the unique possibility to make directed modifications in the mitochondrial genome of yeast, I established a mitochondrial encoded GFP reporter. This reporter allows monitoring of mitochondrial translation with different detection methods and enables more detailed studies focusing on timing and regulation of mitochondrial translation. Furthermore, employing insights gained from bacterial translation, we showed that mitochondrial translation efficiency directly impacts on protein homeostasis of the cytoplasm and lifespan by affecting stress handling. Lastly, we provided first evidence that mitochondrial protein quality control happens at a very early stage directly after or during protein synthesis at the ribosome. Surveillance of protein synthesis and assembly into complexes is important to avoid accumulation of misfolded or unassembled respiratory chain subunits which would disturb mitochondrial function.

In order to thrive in a changing environment all organisms need to ensure protein homeostasis (proteostasis). Proteostasis is ensured by the proteostasis system that monitors the folding status of the proteome and regulates cell physiology and gene expression to counteract any perturbations. An increased burden on the proteostasis system activates Heat shock factor 1 (Hsf1) to induce transcription of the heat shock response (HSR), a transiently induced transcriptional program including core proteostasis genes, importantly those encoding the Hsp70 class of molecular chaperones. The HSR assists cells in counteracting the harmful effects of protein folding stress and restoring proteostasis. The work presented in this thesis is based on experiments with the Saccharomyces cerevisiae (yeast) model with the overall goal of deciphering how Hsp70 detects and impacts on perturbations of cellular proteostasis and controls Hsf1 activity.

In Study I we describe the fundamental mechanism by which Hsp70 maintains Hsf1 in its latent state by controlling its ability to bind DNA. We found that Hsf1 and unfolded proteins directly compete for binding to the Hsp70 substrate-binding domain. During heat shock the pool of unfolded proteins mainly consist of misfolded, newly synthesized proteins. Severe out-titration of Hsp70 by misfolded substrates resulted in unrestrained Hsf1 activity inducing a previously uncharacterized genetic hyper-stress program. More insight into regulation of Hsp70 availability was gained in Study II where the two splice isoforms of the Hsp70 nucleotide exchange factor Fes1 were characterized. We found that the cytosolic splice isoform Fes1S is crucial to release unfolded proteins from Hsp70 and that impaired release results in strong Hsf1 activation.

In Study III we developed methodology to easily measure the rapid changes in Hsf1 activity upon proteostatic perturbations and to monitor protein turnover using the novel bioluminescent reporter NanoLuc optimized for yeast expression (yNluc). In Study IV we report that yNluc also functions as an in vivo reporter that detects severe perturbations of de novo protein folding by its failure to fold to an active conformation under such conditions.

Finally, in Study V we investigated how organellar proteostasis impacts on the availability of cytosolic Hsp70. We found that a lowered mitochondrial proteostatic load as a result of high translation accuracy extended lifespan and improved cytosolic proteostasis capacity, evidenced by more rapid stress recovery and less sensitivity to toxic misfolded proteins. In contrast, lowered mitochondrial translation accuracy decreased lifespan and impaired management of cytosolic protein aggregates as well as elicited a general transcriptional stress response.

Taken together, the findings presented in this thesis advance our understanding of how the regulatory mechanisms of the proteostasis system function. Furthermore, they provide novel methodology that will facilitate future studies to improve our understanding how cells integrate internal and external stress cues to control proteostasis.

Interorganellar connectivity is fundamental for the maintenance of organellar and cellular functionality and viability. This is achieved and maintained by a complex network of signaling cascades, vesicle trafficking between organelles as well as by establishment of direct physical contact at membrane contact sites (MCS). These MCS are sites of close proximity between different organelles, formed by dedicated tethering machineries, that exist between virtually all organelles within a eukaryotic cell. MCS change in size, abundance and molecular architecture in response to metabolic cues and serve to exchange lipids, metabolites and ions. The nucleus-vacuolar junctions (NVJs), establishing contact between the perinuclear ER and the vacuole in yeast, also serve as platform for the biogenesis of a subpopulation of lipid droplets (LD), organelles that function as storage for neutral lipids and contribute to the detoxification of possibly harmful lipid species and aggregated proteins. While it is clear that interorganellar communication at MCS affects cellular functionality at multiple levels, we are just beginning to understand their contribution to cellular protein and lipid homeostasis and their dynamic remodeling in response to metabolic or proteostatic challenges. In Paper I, we identify a novel regulator and component of NVJs, which is essential for contact site formation as well as their expansion in response to glucose exhaustion, controlled by central glucose signaling pathways. In Paper II, we further characterize the role of this protein in ER protein homeostasis and establish it as a transmembrane chaperone that supports the biogenesis of a subset of ER transmembrane proteins, including Nvj1, the main tether of the NVJs, and several enzymes critical for lipid metabolism. Lack of this chaperone leads to aggregation and premature degradation of its substrates, resulting in severe proteostatic and lipid bilayer stress. In Paper III, we investigate the impact of different nutritional regimes on LD biogenesis, subcellular organization and utilization. While the LD subpopulation synthesized at and clustered around the NVJs seems dispensable for long-term survival, we find that a general increase in the synthesis of neutral lipids to be stored in LDs is essential to sustain viability in phosphate-restricted conditions and supports regrowth when de novo fatty acid synthesis is blocked.  In Paper IV, we address interorganellar communication between the mitochondria and the nucleus, showing how mitochondrial translation accuracy modulates nuclear gene expression and affects cytosolic protein homeostasis as well as cellular survival during aging. Collectively, these studies provide new insights into different aspects of organellar communication and their impact on cellular fitness.