Transmembrane proteins represent almost one third of the total cellular proteome, and the majority of them requires translocation into the endoplasmic reticulum (ER) to be folded, sorted and transported to their final subcellular destination. High translational rates during exponential cell growth result in a massive influx of immature proteins into the ER, requiring rapid and accurate folding. Upon proteostatic stress, sensed by the unfolded protein response (UPR), cells increase their ER folding capacity via upregulation of chaperones, folding enzymes and lipid synthesis enzymes to expand the ER membrane. Moreover, different branches of the ER-associated degradation (ERAD) pathway detect and degrade misfolded proteins to prevent protein aggregation in the ER. In this thesis, we identified Snd3 as a novel ER transmembrane chaperone in yeast that is required for the stability of a subset of integral ER proteins, affecting membrane contact site formation and lipid homeostasis. Formation and expansion of the nucleus-vacuole junctions (NVJs), which establish close proximity between the perinuclear ER and the vacuole, not only depended on the presence of Snd3 but also contributed to its spatial organisation within the cell. This chaperone was essential for the stability of the main NVJ tethering protein Nvj1 and in addition concentrated at the NVJs upon glucose exhaustion. Glucose re-addition triggered its rapid disassociation from the NVJs, linking cellular metabolism with membrane contact site dynamics (paper I). Fluorescence and electron microscopy combined with proteomics of microsomal fractions and aggregate isolation revealed the importance of Snd3 for the folding and stability of a subset of ER transmembrane proteins besides Nvj1, with severe effects on the cellular lipidome (paper II). Both functional UPR and ERAD were essential for cell viability upon loss of Snd3. In addition, ER membrane expansion to cope with proteostatic stress was compromised in absence of Snd3, which was due to defective retention of the transcriptional repressor Opi1 at the perinuclear ER, resulting in excess nuclear translocation of Opi1 and insufficient expression of genes required for phospholipid synthesis (paper II). Lipidomic analyses not only revealed a decrease of phosphatidic acid, the phospholipid necessary to anchor Opi1 to the ER membrane, but also an accumulation of very long chain fatty acids (VLCFAs), resulting in overproduction of sphingolipids. Limiting VLCFA synthesis by genetic inactivation of the elongase Elo2 restored contact site formation via stabilisation of Nvj1 and prevented premature cell death (paper III). In sum, we identified a novel molecular chaperone that is required for proper folding of a subset of ER transmembrane proteins, including Nvj1, and thus is critical for efficient membrane contact site formation. When cells exit cell cycle and enter stationary phase due to glucose exhaustion, Snd3 is recruited to the NVJs, where it is stored until glucose availability supports regrowth and the need for folding capacity within the ER increases. Thus, protein targeting to the NVJs contributes to the coordination of cellular folding capacity and lipid metabolism in response to metabolic cues.

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.

Adaptation to fluctuating environments is critical for cellular fitness and survival. Cells need to sense changes in their surroundings and quickly adjust cellular homeostasis and metabolism accordingly. Diverse sensing and signaling pathways govern the cellular responses to extracellular cues, enabling for instance adaptation to changes in nutrient availability. Eukaryotic cells have evolved complex regulatory networks to deal with variable nutrient supply, and most of these evolutionary conserved nutrient signaling pathways are intimately linked to aging and age-associated diseases. Despite significant advances, the adaptation to environmental changes through nutrient signaling pathways and the impact of these pathways on a cell’s lifespan remain incompletely understood. This thesis focuses on the molecular mechanisms by which nutrient depletion affects cellular remodeling and lifespan of the budding yeast Saccharomyces cerevisiae. In paper I, we elucidate the impact of nutritional regimes on cellular survival during aging, with a particular focus on phosphate restriction. We show that phosphate restriction results in an activation of autophagy, the main cellular bulk degradation process, and a prominent extension of lifespan. Our results indicate that longevity induced by phosphate restriction relies on the sequential and coordinated function of autophagy and the multivesicular body pathway, a catabolic process critical for the degradation of plasma membrane components. In addition, we find the nutrient-responsive kinase Pho85 to be essential for autophagy induction and cellular fitness upon phosphate restriction. In paper II, we illustrate how nutrition limitation affects interorganellar communication as well as lipid droplet biogenesis, subcellular organization and utilization. We demonstrate that phosphate depletion induces the remodeling and expansion of the contacts between the perinuclear ER and the vacuole, the so-called nucleus-vacuole junctions. Moreover, we show that the biosynthesis of sterol esters, which are stored in lipid droplets upon nutrient depletion, is essential for survival upon phosphate but not glucose exhaustion. In paper III, we identify Snd3 as novel component of the nucleus-vacuole junctions, critical for membrane contact site formation and dynamic remodeling upon glucose exhaustion. Additionally, we show that the regulatory function of Snd3 is governed by central glucose signaling pathways. Taken together, our studies advance our understanding of how nutritional regimes and signaling pathways impact on cellular remodeling and survival during aging.