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.

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.

Metals play a crucial role in cellular biology. Bulk and trace metals such as calcium and manganese regulate a plethora of cellular processes ranging from signaling and oxidative stress to proteostasis and energy metabolism. Fine-tuning metal levels and distribution safeguards all forms of life from compromised cellular fitness and cell death elicited by metal deficiency or overload. However, the underlying molecular mechanisms eventually leading to cellular demise remain elusive. In this thesis, we studied the fundamental impact of disrupted metal homeostasis on cellular survival focusing on mitochondrial and lysosomal processes in Saccharomyces cerevisiae and Drosophila melanogaster. In Paper I, we establish Coenzyme Q (CoQ) biosynthesis in mitochondria as the prime target of cellular manganese overload and propose a molecular mechanism underlying manganese toxicity. Combining proteomics, genome-wide screening and comprehensive metal analyses, we identify mismetallation of the di-iron hydroxylase Coq7, an enzyme of CoQ biosynthesis, as cause for the CoQ deficiency upon manganese overload. Overexpression of Coq7 not only restored CoQ-mediated electron transport through the respiratory chain but also prevented age-associated death. Expanding from trace to bulk metals, we further assessed the impact of disrupted calcium and manganese homeostasis on cellular survival. In Paper II, we created a fluorescence-based reporter system for the Ca²⁺/calmodulin-dependent phosphatase calcineurin, a nexus for cell stress-induced signaling. Combining our reporters with a live/dead staining allows for quantification of acute and chronic changes in calcium signaling in living, unperturbed cells. In Paper III, we elucidate the impact of nutritional regimes known to improve cellular survival on cells compromised in the handling of calcium and manganese due to the absence of Pmr1, a Ca²⁺/Mn²⁺ ATPase of the secretory pathway. We demonstrate that caloric restriction prevents manganese-induced disruption of mitochondrial energy metabolism and improves survival independent of calcineurin activity and CoQ biosynthesis. In Papers IV and V, we studied the interplay of metal levels and calcium signaling in the context of neurodegeneration and report that calcineurin stimulates lysosomal proteolysis, thereby preventing proteotoxicity in yeast and Drosophila models for Parkinson’s disease. Collectively, our results provide new insights into the consequences of disrupted metal homeostasis for cellular fitness and unravel a novel link between manganese overload, mitochondrial bioenergetics and CoQ biosynthesis conserved across species.

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.

Respiratory chains are crucial for cellular energy conversion and consist of multi-subunit complexes that can assemble into supercomplexes. These structures have been intensively characterized in various organisms, but their physiological roles remain unclear. Here, we elucidate their function by leveraging a high-resolution structural model of yeast respiratory supercomplexes that allowed us to inhibit supercomplex formation by mutation of key residues in the interaction interface. Analyses of a mutant defective in supercomplex formation, which still contains fully functional individual complexes, show that the lack of supercomplex assembly delays the diffusion of cytochrome c between the separated complexes, thus reducing electron transfer efficiency. Consequently, competitive cellular fitness is severely reduced in the absence of supercomplex formation and can be restored by overexpression of cytochrome c. In sum, our results establish how respiratory supercomplexes increase the efficiency of cellular energy conversion, thereby providing an evolutionary advantage for aerobic organisms.

The protein phosphatase calcineurin is activated in response to rising intracellular Ca2+ levels and impacts fundamental cellular processes in organisms ranging from yeast to humans. In fungi, calcineurin orchestrates cellular adaptation to diverse environmental challenges and is essential for virulence of pathogenic species. To enable rapid and large-scale assessment of calcineurin activity in living, unperturbed yeast cells, we have generated stable and destabilized GFP transcriptional reporters under the control of a calcineurin-dependent response element (CDRE). Using the reporters, we show that the rapid dynamics of calcineurin activation and deactivation can be followed by flow cytometry and fluorescence microscopy. This system is compatible with live/dead staining that excludes confounding dead cells from the analysis. The reporters provide technology to monitor calcineurin dynamics during stress and ageing and may serve as a drug-screening platform to identify novel antifungal compounds that selectively target calcineurin.

The loss of lipid homeostasis can lead to lipid overload and is associated with a variety of disease states. However, little is known as to how the disruption of lipid regulation or lipid overload affects cell survival. In this study we investigated how excess diacylglycerol (DG), a cardinal metabolite suspected to mediate lipotoxicity, compromises the survival of yeast cells. We reveal that increased DG achieved by either genetic manipulation or pharmacological administration of 1,2-dioctanoyls-n-glycerol (DOG) triggers necrotic cell death. The toxic effects of DG are linked to glucose metabolism and require a functional Rim101 signaling cascade involving the Rim21-dependent sensing complex and the activation of a calpain-like protease. The Rim101 cascade is an established pathway that triggers a transcriptional response to alkaline or lipid stress. We propose that the Rim101 pathway senses DG-induced lipid perturbation and conducts a signaling response that either facilitates cellular adaptation or triggers lipotoxic cell death. Using established models of lipotoxicity, i.e., high-fat diet in Drosophila and palmitic acid administration in cultured human endothelial cells, we present evidence that the core mechanism underlying this calpain-dependent lipotoxic cell death pathway is phylogenetically conserved.

Mitochondrial dysfunction is a common feature of many neurodegenerative diseases, including proteinopathies such as Alzheimer's or Parkinson's disease, which are characterized by the deposition of aggregated proteins in the form of insoluble fibrils or plaques. The distinct molecular processes that eventually result in mitochondrial dysfunction during neurodegeneration are well studied but still not fully understood. However, defects in mitochondrial fission and fusion, mitophagy, oxidative phosphorylation and mitochondrial bioenergetics have been linked to cellular demise. These processes are influenced by the lipid environment within mitochondrial membranes as, besides membrane structure and curvature, recruitment and activity of different proteins also largely depend on the respective lipid composition. Hence, the interaction of neurotoxic proteins with certain lipids and the modification of lipid composition in different cell compartments, in particular mitochondria, decisively impact cell death associated with neurodegeneration. Here, we discuss the relevance of mitochondrial lipids in the pathological alterations that result in neuronal demise, focussing on proteinopathies.

The degeneration of dopaminergic neurons during Parkinson's disease (PD) is intimately linked to malfunction of alpha-synuclein (alpha Syn), the main component of the proteinaceous intracellular inclusions characteristic for this pathology. The cytotoxicity of alpha Syn has been attributed to disturbances in several biological processes conserved from yeast to humans, including Ca2+ homeostasis, general lysosomal function and autophagy. However, the precise sequence of events that eventually results in cell death remains unclear. Here, we establish a connection between the major lysosomal protease cathepsin D (CatD) and the Ca2+/calmodulin-dependent phosphatase calcineurin. In a yeast model for PD, high levels of human alpha Syn triggered cytosolic acidification and reduced vacuolar hydrolytic capacity, finally leading to cell death. This could be counteracted by overexpression of yeast CatD (Pep4), which re-installed pH homeostasis and vacuolar proteolytic function, decreased alpha Syn oligomers and aggregates, and provided cytoprotection. Interestingly, these beneficial effects of Pep4 were independent of autophagy. Instead, they required functional calcineurin signaling, since deletion of calcineurin strongly reduced both the proteolytic activity of endogenous Pep4 and the cytoprotective capacity of overexpressed Pep4. Calcineurin contributed to proper endosomal targeting of Pep4 to the vacuole and the recycling of the Pep4 sorting receptor Pep1 from prevacuolar compartments back to the trans-Golgi network. Altogether, we demonstrate that stimulation of this novel calcineurin-Pep4 axis reduces alpha Syn cytotoxicity.