Lipid droplets (LDs) are fat storage organelles critical for energy and lipid metabolism. Upon nutrient exhaustion, cells consume LDs via gradual lipolysis or via lipophagy, the en bloc uptake of LDs into the vacuole. Here, we show that LDs dock to the vacuolar membrane via a contact site that is required for lipophagy in yeast. The LD-localized LDO proteins carry an intrinsically disordered region that directly binds vacuolar Vac8 to form vCLIP, the vacuolar-LD contact site. Nutrient limitation drives vCLIP formation, and its inactivation blocks lipophagy, resulting in impaired caloric restriction-induced longevity. We establish a functional link between lipophagy and microautophagy of the nucleus, both requiring Vac8 to form respective contact sites upon metabolic stress. In sum, we identify the tethering machinery of vCLIP and find that Vac8 provides a platform for multiple and competing contact sites associated with autophagy.

Overexposure to manganese disrupts cellular energy metabolism across species, but the molecular mechanism underlying manganese toxicity remains enigmatic. Here, we report that excess cellular manganese selectively disrupts coenzyme Q (CoQ) biosynthesis, resulting in failure of mitochondrial bioenergetics. While respiratory chain complexes remain intact, the lack of CoQ as lipophilic electron carrier precludes oxidative phosphorylation and leads to premature cell and organismal death. At a molecular level, manganese overload causes mismetallation and proteolytic degradation of Coq7, a diiron hydroxylase that catalyzes the penultimate step in CoQ biosynthesis. Coq7 overexpression or supplementation with a CoQ headgroup analog that bypasses Coq7 function fully corrects electron transport, thus restoring respiration and viability. We uncover a unique sensitivity of a diiron enzyme to mismetallation and define the molecular mechanism for manganese-induced bioenergetic failure that is conserved across species.

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.

The capacity of a cell to maintain proteostasis progressively declines during aging. Virtually all age-associated neurodegenerative disorders associated with aggregation of neurotoxic proteins are linked to defects in the cellular proteostasis network, including insufficient lysosomal hydrolysis. Here, we report that proteotoxicity in yeast and Drosophila models for Parkinson’s disease can be prevented by increasing the bioavailability of Ca2+, which adjusts intracellular Ca2+ handling and boosts lysosomal proteolysis. Heterologous expression of human α-synuclein (αSyn), a protein critically linked to Parkinson’s disease, selectively increases total cellular Ca2+ content, while the levels of manganese and iron remain unchanged. Disrupted Ca2+ homeostasis results in inhibition of the lysosomal protease cathepsin D and triggers premature cellular and organismal death. External administration of Ca2+ reduces αSyn oligomerization, stimulates cathepsin D activity and in consequence restores survival, which critically depends on the Ca2+/calmodulin-dependent phosphatase calcineurin. In flies, increasing the availability of Ca2+ discloses a neuroprotective role of αSyn upon manganese overload. In sum, we establish a molecular interplay between cathepsin D and calcineurin that can be activated by Ca2+ administration to counteract αSyn proteotoxicity.

Mitochondrial defects can cause a variety of human diseases and protective mechanisms exist to maintain mitochondrial functionality. Imbalances in mitochondrial proteostasis trigger a transcriptional program, termed mitochondrial unfolded protein response (mtUPR). However, the temporal sequence of events in mtUPR is unclear and the consequences on mitochondrial protein import are controversial. Here, we have quantitatively analyzed all main import pathways into mitochondria after different time spans of mtUPR induction. Kinetic analyses reveal that protein import into all mitochondrial subcompartments strongly increases early upon mtUPR and that this is accompanied by rapid remodelling of the mitochondrial signature lipid cardiolipin. Genetic inactivation of cardiolipin synthesis precluded stimulation of protein import and compromised cellular fitness. At late stages of mtUPR upon sustained stress, mitochondrial protein import efficiency declined. Our work clarifies the enigma of protein import upon mtUPR and identifies sequential mtUPR stages, in which an early increase in protein biogenesis to restore mitochondrial proteostasis is followed by late stages characterized by a decrease in import capacity upon prolonged stress induction.

Nutrient limitation results in an activation of autophagy in organisms ranging from yeast, nematodes and flies to mammals. Several evolutionary conserved nutrient-sensing kinases are critical for efficient adaptation of yeast cells to glucose, nitrogen or phosphate depletion, subsequent cell-cycle exit and the regulation of autophagy. Here, we demonstrate that phosphate restriction results in a prominent extension of yeast lifespan that requires the coordinated activity of autophagy and the multivesicular body pathway, enabling efficient turnover of cytoplasmic and plasma membrane cargo. While the multivesicular body pathway was essential during the early days of aging, autophagy contributed to long-term survival at later days. The cyclin-dependent kinase Pho85 was critical for phosphate restriction-induced autophagy and full lifespan extension. In contrast, when cell-cycle exit was triggered by exhaustion of glucose instead of phosphate, Pho85 and its cyclin, Pho80, functioned as negative regulators of autophagy and lifespan. The storage of phosphate in form of polyphosphate was completely dispensable to in sustaining viability under phosphate restriction. Collectively, our results identify the multifunctional, nutrient-sensing kinase Pho85 as critical modulator of longevity that differentially coordinates the autophagic response to distinct kinds of starvation.

Autophagy is a cellular recycling program which efficiently reduces the cellular burden of ageing. Autophagy is characterised by nucleation of isolation membranes, which grow in size and further expand to form autophagosomes, engulfing cellular material to be degraded by fusion with lysosomes (vacuole in yeast). Autophagosomal membranes do not bud from a single cell organelle, but are generated de novo. Several lipid sources for autophagosomal membranes have been identified, but the whole process of their generation is complex and not entirely understood. In this study, we investigated how the mitochondrial outer membrane protein porin 1 (Por1), the yeast orthologue of mammalian voltage-dependent anion channel (VDAC), affects autophagy in yeast. We show that POR1 deficiency reduces the autophagic capacity and leads to changes in vacuole and lipid homeostasis. We further investigated whether limited phosphatidylethanolamine (PE) availability in por1∆ was causative for reduced autophagy by overexpression of the PE-generating phosphatidylserine decarboxylase 1 (Psd1). Altogether, our results show that POR1 deficiency is associated with reduced autophagy, which can be circumvented by additional PSD1 overexpression. This suggests a role for Por1 in Psd1-mediated autophagy regulation.

Membrane contact sites facilitate the exchange of metabolites between organelles to support interorganellar communication. The nucleus-vacuole junctions (NVJs) establish physical contact between the perinuclear endoplasmic reticulum (ER) and the vacuole. Although the NVJ tethers are known, how NVJ abundance and composition are controlled in response to metabolic cues remains elusive. Here, we identify the ER protein Snd3 as central factor for NVJ formation. Snd3 interacts with NVJ tethers, supports their targeting to the contacts, and is essential for NVJ formation. Upon glucose exhaustion, Snd3 relocalizes from the ER to NVJs and promotes contact expansion regulated by central glucose signaling pathways. Glucose replenishment induces the rapid dissociation of Snd3 from the NVJs, preceding the slow disassembly of the junctions. In sum, this study identifies a key factor required for formation and regulation of NVJs and provides a paradigm for metabolic control of membrane contact sites.

Recently, we reported that, in mice, hunger causes the autophagy-dependent release of a protein called acyl-CoA-binding protein or diazepam binding inhibitor (ACBP/DBI) from cells, resulting in an increase in plasma ACBP concentrations. Administration of extra ACBP is orexigenic and obesogenic, while its neutralization is anorexigenic in mice, suggesting that ACBP is a major stimulator of appetite and lipo-anabolism. Accordingly, obese persons have higher circulating ACBP levels than lean individuals, and anorexia nervosa is associated with subnormal ACBP plasma concentrations. Here, we investigated whether ACBP might play a phylogenetically conserved role in appetite stimulation. We found that extracellular ACBP favors sporulation in Saccharomyces cerevisiae, knowing that sporulation is a strategy for yeast to seek new food sources. Moreover, in the nematode Caenorhabditis elegans, ACBP increased the ingestion of bacteria as well as the frequency pharyngeal pumping. These observations indicate that ACBP has a phylogenetically ancient role as a 'hunger factor' that favors food intake.

The mitochondrial proteome is built mainly by import of nuclear-encoded precursors, which are targeted mostly by cleavable presequences. Presequence processing upon import is essential for proteostasis and survival, but the consequences of dysfunctional protein maturation are unknown. We find that impaired presequence processing causes accumulation of precursors inside mitochondria that form aggregates, which escape degradation and unexpectedly do not cause cell death. Instead, cells survive via activation of a mitochondrial unfolded protein response (mtUPR)-like pathway that is triggered very early after precursor accumulation. In contrast to classical stress pathways, this immediate response maintains mitochondrial protein import, membrane potential, and translation through translocation of the nuclear HMG-box transcription factor Roxl to mitochondria. Roxl binds mtDNA and performs a TFAM-like function pivotal for transcription and translation. Induction of early mtUPR provides a reversible stress model to mechanistically dissect the initial steps in mtUPR pathways with the stressTFAM Roxl as the first line of defense.