Nutrient starvation initiates cell cycle exit and entry into quiescence, a reversible, non-proliferative state characterized by stress tolerance, longevity and large-scale remodeling of subcellular structures. Depending on the nature of the depleted nutrient, yeast cells are assumed to enter heterogeneous quiescent states with unique but mostly unexplored characteristics. Here, we show that storage and consumption of neutral lipids in lipid droplets (LDs) differentially impacts the regulation of quiescence driven by glucose or phosphate starvation. Upon prolonged glucose exhaustion, LDs were degraded in the vacuole via Atg1-dependent lipophagy. In contrast, yeast cells entering quiescence due to phosphate exhaustion massively over-accumulated LDs that clustered at the vacuolar surface but were not engulfed via lipophagy. Excessive LD biogenesis required contact formation between the endoplasmic reticulum and the vacuole at nucleus-vacuole junctions and was accompanied by a shift of the cellular lipid profile from membrane towards storage lipids, driven by a transcriptional upregulation of enzymes generating neutral lipids, in particular sterol esters. Importantly, sterol ester biogenesis was critical for long-term survival of phosphate-exhausted cells and supported rapid quiescence exit upon nutrient replenishment, but was dispensable for survival and regrowth of glucose-exhausted cells. Instead, these cells relied on de novo synthesis of sterols and fatty acids for quiescence exit and regrowth. Phosphate-exhausted cells efficiently mobilized storage lipids to support several rounds of cell division even in presence of inhibitors of fatty acid and sterol biosynthesis. In sum, our results show that neutral lipid biosynthesis and mobilization to support quiescence maintenance and exit is tailored to the respective nutrient scarcity.

Eukaryotic membranes are compartmentalized into distinct micro- and nanodomains that rearrange dynamically in response to external and internal cues. This lateral heterogeneity of the lipid bilayer and associated clustering of distinct membrane proteins contribute to the spatial organization of numerous cellular processes. Here, we show that membrane microdomains within the endoplasmic reticulum (ER) of yeast cells are reorganized during metabolic reprogramming and aging. Using biosensors with varying transmembrane domain length to map lipid bilayer thickness, we demonstrate that in young cells, microdomains of increased thickness mainly exist within the nuclear ER, while progressing cellular age drives the formation of numerous microdomains specifically in the cortical ER. Partitioning of biosensors with long transmembrane domains into these microdomains increased protein stability and prevented autophagic removal. In contrast, reporters with short transmembrane domains progressively accumulated at the membrane contact site between the nuclear ER and the vacuole, the so-called nucleus-vacuole junction (NVJ), and were subjected to turnover via selective microautophagy occurring specifically at these sites. Reporters with long transmembrane domains were excluded from the NVJ. Our data reveal age-dependent rearrangement of the lateral organization of the ER and establish transmembrane domain length as a determinant of membrane contact site localization and autophagic degradation.