Amino acids trigger MDC-dependent mitochondrial remodeling by altering mitochondrial function.

Cells utilize numerous pathways to maintain mitochondrial homeostasis, including a recently identified mechanism that adjusts the content of the outer mitochondrial membrane (OMM) through formation of OMM-derived multilamellar domains called mitochondrial-derived compartments, or MDCs. MDCs are triggered by perturbations in mitochondrial lipid and protein content, as well as increases in intracellular amino acids. Here, we sought to understand how amino acids trigger MDCs. We show that amino acid-activation of MDCs is dependent on the functional state of mitochondria. While amino acid excess triggers MDC formation when cells are grown on fermentable carbon sources, stimulating mitochondrial biogenesis blocks MDC formation. Moreover, amino acid elevation depletes TCA cycle metabolites in yeast, and preventing consumption of TCA cycle intermediates for amino acid catabolism suppresses MDC formation. Finally, we show that directly impairing the TCA cycle is sufficient to trigger MDC formation in the absence of amino acid stress. These results demonstrate that amino acids stimulate MDC formation by perturbing mitochondrial metabolism.

The phospholipids cardiolipin and phosphatidylethanolamine differentially regulate MDC biogenesis.

Cells utilize multiple mechanisms to maintain mitochondrial homeostasis. We recently characterized a pathway that remodels mitochondria in response to metabolic alterations and protein overload stress. This remodeling occurs via the formation of large membranous structures from the mitochondrial outer membrane called mitochondrial-derived compartments (MDCs), which are eventually released from mitochondria and degraded. Here, we conducted a microscopy-based screen in budding yeast to identify factors that regulate MDC formation. We found that two phospholipids, cardiolipin (CL) and phosphatidylethanolamine (PE), differentially regulate MDC biogenesis. CL depletion impairs MDC biogenesis, whereas blocking mitochondrial PE production leads to constitutive MDC formation. Additionally, in response to metabolic MDC activators, cellular and mitochondrial PE declines, and overexpressing mitochondrial PE synthesis enzymes suppress MDC biogenesis. Altogether, our data indicate a requirement for CL in MDC biogenesis and suggest that PE depletion may stimulate MDC formation downstream of MDC-inducing metabolic stress.

Sexual dimorphism in the gonad lipidome of blue mussels (Mytilus sp.): New insights from a global lipidomics approach.

Blue mussels (Mytilus sp.) are an economically important species for European aquaculture. Their importance as a food source is expected to increase in the coming net-zero society due to their low environmental footprint; however, their production is affected by anthropogenic stressors and climate change. During reproduction, lipids are key molecules for mussels as they are the main source of energy on which newly hatched embryos depend in the first days of their development. In this work, blue mussels of different origins are analysed, focusing on the differences in lipid composition between the ovary (BMO) and the testis (BMT). The lipidome of blue mussel gonads (BMG) is studied here by combining traditional lipid profiling methods, such as fatty acid and lipid class analysis, with untargeted liquid chromatography-mass spectrometry (LC-MS) lipidomics. The approach used here enabled the identification of 770 lipid molecules from 23 different lipid classes in BMG. BMT, which consists of billions of spermatocytes, had greater amounts of cell membrane and membrane lipid components such as FA18:0, C20 polyunsaturated fatty acids (PUFA), free sterols (ST), ceramide phosphoethanolamines (CerPE), ceramide aminoethylphosphonates (CAEP), cardiolipins (CL), glycerophosphocholines (PC), glycerophosphoethanolamines (PE) and glycerophosphoserines (PS). In BMO, saturated fatty acids (FA14:0 and FA16:0), monounsaturated fatty acids (MUFA) and other storage components such as C18-PUFA accumulated in triradylglycerolipids (TG) and alkyldiacylglycerols (neutral plasmalogens, TG O-), which, together with terpenes, wax esters and cholesterol esters, make up most of oocytes yolk reserves. BMO also had higher levels of ceramides (Cer) and generally alkyl/alkenyl glycerophospholipids (mainly plasmanyl/plasmenyl PC), suggesting a role for these lipids in vitellogenesis. Non-methylene interrupted dienoic fatty acids (NMID FA), typically found in plasmalogens, were the only membrane-forming PUFA predominantly detected in BMO. The results of this study are of great importance for clarifying the lipid composition of BMG and provide an important basis for future studies on the reproductive physiology of these organisms.

Metabolism and Excretion of Therapeutic Peptides: Current Industry Practices, Perspectives, and Recommendations.

Therapeutic peptides (TPeps) have expanded from the initial endogenous peptides to complex modified peptides through medicinal chemistry efforts for almost a century. Different from small molecules and large proteins, the diverse submodalities of TPeps have distinct structures and carry different absorption, distribution, metabolism, and excretion (ADME) properties. There is no distinct regulatory guidance for the industry on conducting ADME studies (what, how, and when) for TPeps. Therefore, the Peptide ADME Working Group sponsored by the Translational and ADME Sciences Leadership Group of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) was formed with the goal to develop a white paper focusing on metabolism and excretion studies to support discovery and development of TPeps. In this paper, the key learnings from an IQ industry survey and U.S. Food and Drug Administration/European Medicines Agency submission documents of TPeps approved between 2011 and 2022 are outlined in detail. In addition, a comprehensive assessment of in vitro and in vivo metabolism and excretion studies, mitigation strategies for TPep metabolism, analytical tools to conduct studies, regulatory status, and Metabolites in Safety Testing considerations are provided. Finally, an industry recommendation on conducting metabolism and excretion studies is proposed for regulatory filing of TPeps. SIGNIFICANCE STATEMENT: This white paper presents current industry practices for metabolism and excretion studies of therapeutic peptides based on an industry survey, regulatory submission documents, and expert opinions from the participants in the Peptide Absorption, Distribution, Metabolism, and Excretion Working Group of the International Consortium for Innovation and Quality in Pharmaceutical Development. The group also provides recommendations on the Metabolites in Safety Testing considerations and metabolism and excretion studies for regulatory filing of therapeutic peptides.

Mitochondrial-Derived Compartments are Multilamellar Domains that Encase Membrane Cargo and Cytosol.

Preserving the health of the mitochondrial network is critical to cell viability and longevity. To do so, mitochondria employ several membrane remodeling mechanisms, including the formation of mitochondrial-derived vesicles (MDVs) and compartments (MDCs) to selectively remove portions of the organelle. In contrast to well-characterized MDVs, the distinguishing features of MDC formation and composition remain unclear. Here we used electron tomography to observe that MDCs form as large, multilamellar domains that generate concentric spherical compartments emerging from mitochondrial tubules at ER-mitochondria contact sites. Time-lapse fluorescence microscopy of MDC biogenesis revealed that mitochondrial membrane extensions repeatedly elongate, coalesce, and invaginate to form these compartments that encase multiple layers of membrane. As such, MDCs strongly sequester portions of the outer mitochondrial membrane, securing membrane cargo into a protected domain, while also enclosing cytosolic material within the MDC lumen. Collectively, our results provide a model for MDC formation and describe key features that distinguish MDCs from other previously identified mitochondrial structures and cargo-sorting domains.

Reasons for admission to a general medical hospital for patients taking clozapine.

Background: Clozapine is associated with a diverse range of side effects. In addition, patients prescribed clozapine commonly suffer with medical comorbidities. Objectives: This study aimed to characterise patients prescribed clozapine who required medical admission, understand reasons for admission, identify areas for interventions to prevent future admission and describe clozapine management during the inpatient stay. Design: We conducted a retrospective analysis of patients prescribed clozapine who were admitted to a general medical hospital in a 12-month period. Method: Data were collected using electronic drug charts and notes. Results: In total, 114 clozapine patients were hospitalised. Twenty-eight patients (25%) were admitted because of infection, 12 (11%) were elective admissions and 12 (11%) had gastrointestinal problems. Most patients admitted were Black (54%) and half were female. Few changes were made to clozapine dosing on admission or during the inpatient stay. Most patients had been taking clozapine for many years at the point of admission, the majority were able to continue taking it for the duration of their medical treatment and were discharged on the same dose they were taking prior to admission. Clozapine plasma concentrations were not consistently measured with only 18 (16%) patients having one or more plasma concentrations determined during their admission. The median clozapine plasma concentration on admission was 0.48 mg/L (nor-clozapine 0.21 mg/L), with a range of 0.09 to 3.9 mg/L. Three patients were admitted to the intensive care unit during their admission; all were discharged on clozapine. Four patients died; one from lung adenocarcinoma, one bowel obstruction, one cardiac arrest and one chest sepsis. In total, 27 patients (23%) had their clozapine stopped on admission, 6 (22% of this group) unintentionally. Conclusions: Our study found that the most common reason for admission for patients taking clozapine was infection. Plasma concentrations were not measured routinely despite clozapine having a narrow therapeutic index and enhanced potential for toxicity in the medically unwell patient.

Fission yeast Dap1 heme iron-coordinating residue Y83 is required for cytochromes P450 function.

Fission yeast Dap1 is a heme binding protein required for cytochromes P450 activity. Here, we tested whether Dap1 axial coordination of heme iron is required for its role in the function of the cytochrome P450 enzymes, Erg5 and Erg11. Two different dap1 mutants predicted to alter iron coordination failed to rescue growth on cobalt chloride containing medium which requires Erg5 and Erg11. In addition, deletion of dap1 + did not affect expression of Erg5 or Erg11. PGRMC1, a mammalian Dap1 homolog, does not require heme binding to bind and stabilize cytochromes P450. These experiments highlight important functional differences between these conserved proteins.

SPOTting stress at the mitochondrial outer membrane.

Li et al. (2022) discover that Toxoplasma infection triggers remodeling of the mitochondrial outer membrane through generation of a mitochondrial subdomain termed "structure positive for outer mitochondrial membrane" (SPOT).

Parallel evolution of Pseudomonas aeruginosa phage resistance and virulence loss in response to phage treatment in vivo and in vitro.

With rising antibiotic resistance, there has been increasing interest in treating pathogenic bacteria with bacteriophages (phage therapy). One limitation of phage therapy is the ease at which bacteria can evolve resistance. Negative effects of resistance may be mitigated when resistance results in reduced bacterial growth and virulence, or when phage coevolves to overcome resistance. Resistance evolution and its consequences are contingent on the bacteria-phage combination and their environmental context, making therapeutic outcomes hard to predict. One solution might be to conduct 'in vitro evolutionary simulations' using bacteria-phage combinations from the therapeutic context. Overall, our aim was to investigate parallels between in vitro experiments and in vivo dynamics in a human participant. Evolutionary dynamics were similar, with high levels of resistance evolving quickly with limited evidence of phage evolution. Resistant bacteria-evolved in vitro and in vivo-had lower virulence. In vivo, this was linked to lower growth rates of resistant isolates, whereas in vitro phage resistant isolates evolved greater biofilm production. Population sequencing suggests resistance resulted from selection on de novo mutations rather than sorting of existing variants. These results highlight the speed at which phage resistance can evolve in vivo, and how in vitro experiments may give useful insights for clinical evolutionary outcomes.

Lung-restricted ALK5 inhibition avoids systemic toxicities associated with TGFß pathway inhibition.

Systemic therapies targeting transforming growth factor beta (TGFß) or TGFßR1 kinase (ALK5) have been plagued by toxicities including cardiac valvulopathy and bone physeal dysplasia in animals, posing a significant challenge for clinical development in pulmonary indications. The current work aims to demonstrate that systemic ALK5-associated toxicities can be mitigated through localized lung delivery. Lung-selective (THRX-144644) and systemically bioavailable (galunisertib) ALK5 inhibitors were compared to determine whether lung selectivity is sufficient to maintain local tissue concentrations while mitigating systemic exposure and consequent pathway-related findings. Both molecules demonstrated potent ALK5 activity in rat precision cut lung slices (PCLS; p-SMAD3 half-maximal inhibitory concentration [IC50], 141 nM and 1070 nM for THRX-144644 and galunisertib, respectively). In 14-day repeat-dose studies in rats, dose-related cardiac valvulopathy was recapitulated with oral galunisertib at doses ≥150 mg/kg/day. In contrast, inhaled nebulized THRX-144644 did not cause similar systemic findings up to the maximally tolerated doses in rats or dogs (10 and 1.5 mg/kg/day, respectively). THRX-144644 lung-to-plasma ratios ranged from 100- to 1200-fold in rats and dogs across dose levels. THRX-144644 lung trough (24 h) concentrations in rats and dogs ranged from 3- to 17-fold above the PCLS IC50 across tolerated doses. At a dose level exceeding tolerability (60 mg/kg/day; 76-fold above PCLS IC50) minimal heart and bone changes were observed when systemic drug concentrations reached pharmacologic levels. In conclusion, the current preclinical work demonstrates that localized pulmonary delivery of an ALK5 inhibitor leads to favorable TGFß pathway pharmacodynamic inhibition in lung while minimizing key systemic toxicities.

Peptide transport in Bacillus subtilis - structure and specificity in the extracellular solute binding proteins OppA and DppE.

Peptide transporters play important nutritional and cell signalling roles in Bacillus subtilis, which are pronounced during stationary phase adaptations and development. Three high-affinity ATP-binding cassette (ABC) family transporters are involved in peptide uptake - the oligopeptide permease (Opp), another peptide permease (App) and a less well-characterized dipeptide permease (Dpp). Here we report crystal structures of the extracellular substrate binding proteins, OppA and DppE, which serve the Opp and Dpp systems, respectively. The structure of OppA was determined in complex with endogenous peptides, modelled as Ser-Asn-Ser-Ser, and with the sporulation-promoting peptide Ser-Arg-Asn-Val-Thr, which bind with K d values of 0.4 and 2 µM, respectively, as measured by isothermal titration calorimetry. Differential scanning fluorescence experiments with a wider panel of ligands showed that OppA has highest affinity for tetra- and penta-peptides. The structure of DppE revealed the unexpected presence of a murein tripeptide (MTP) ligand, l-Ala-d-Glu-meso-DAP, in the peptide binding groove. The mode of MTP binding in DppE is different to that observed in the murein peptide binding protein, MppA, from Escherichia coli, suggesting independent evolution of these proteins from an OppA-like precursor. The presence of MTP in DppE points to a role for Dpp in the uptake and recycling of cell wall peptides, a conclusion that is supported by analysis of the genomic context of dpp, which revealed adjacent genes encoding enzymes involved in muropeptide catabolism in a gene organization that is widely conserved in Firmicutes.

Mitochondrial-derived compartments facilitate cellular adaptation to amino acid stress.

Amino acids are essential building blocks of life. However, increasing evidence suggests that elevated amino acids cause cellular toxicity associated with numerous metabolic disorders. How cells cope with elevated amino acids remains poorly understood. Here, we show that a previously identified cellular structure, the mitochondrial-derived compartment (MDC), functions to protect cells from amino acid stress. In response to amino acid elevation, MDCs are generated from mitochondria, where they selectively sequester and deplete SLC25A nutrient carriers and their associated import receptor Tom70 from the organelle. Generation of MDCs promotes amino acid catabolism, and their formation occurs simultaneously with transporter removal at the plasma membrane via the multivesicular body (MVB) pathway. The combined loss of vacuolar amino acid storage, MVBs, and MDCs renders cells sensitive to high amino acid stress. Thus, we propose that MDCs operate as part of a coordinated cell network that facilitates amino acid homeostasis through post-translational nutrient transporter remodeling.

A nuclear-based quality control pathway for non-imported mitochondrial proteins.

Mitochondrial import deficiency causes cellular toxicity due to the accumulation of non-imported mitochondrial precursor proteins, termed mitoprotein-induced stress. Despite the burden mis-localized mitochondrial precursors place on cells, our understanding of the systems that dispose of these proteins is incomplete. Here, we cataloged the location and steady-state abundance of mitochondrial precursor proteins during mitochondrial impairment in Saccharomyces cerevisiae. We found that a number of non-imported mitochondrial proteins localize to the nucleus, where they are subjected to proteasome-dependent degradation through a process we term nuclear-associated mitoprotein degradation (mitoNUC). Recognition and destruction of mitochondrial precursors by the mitoNUC pathway requires the presence of an N-terminal mitochondrial targeting sequence and is mediated by combined action of the E3 ubiquitin ligases San1, Ubr1, and Doa10. Impaired breakdown of precursors leads to alternative sequestration in nuclear-associated foci. These results identify the nucleus as an important destination for the disposal of non-imported mitochondrial precursors.

ER targeting of non-imported mitochondrial carrier proteins is dependent on the GET pathway.

Deficiencies in mitochondrial import cause the toxic accumulation of non-imported mitochondrial precursor proteins. Numerous fates for non-imported mitochondrial precursors have been identified in budding yeast, including proteasomal destruction, deposition into protein aggregates, and mistargeting to other organelles. Amongst organelles, the ER has emerged as a key destination for a subset of non-imported mitochondrial proteins. However, how ER targeting of various types of mitochondrial proteins is achieved remains incompletely understood. Here, we show that the ER delivery of endogenous mitochondrial transmembrane proteins, especially those belonging to the SLC25A mitochondrial carrier family, is dependent on the guided entry of tail-anchored proteins (GET) complex. Without a functional GET pathway, non-imported mitochondrial proteins destined for the ER are alternatively sequestered into Hsp42-dependent protein foci. Loss of the GET pathway is detrimental to yeast cells experiencing mitochondrial import failure and prevents re-import of mitochondrial proteins from the ER via the ER-SURF pathway. Overall, this study outlines an important role for the GET complex in ER targeting of non-imported mitochondrial carrier proteins.

Comparing the effectiveness of twine- and binder-seeding in the Laminariales species Alaria esculenta and Saccharina latissima.

The continuing expansion of seaweed cultivation could assist in ensuring future global food security. The Laminariales species Alaria esculenta and Saccharina latissima are each cultivated for food across their European ranges. The predominant method for cultivating European kelps involves growing juveniles on twine within a hatchery which is then deployed at a farm site. The associated hatchery and deployment cost of this approach are relatively high. A new and innovative methodology-called binder-seeding-can reduce these costs, but, has yet to be validated. We compare the biomass yield and morphology of A. esculenta and S. latissima cultured using either the traditional twine-longline method or binder-seeding onto AlgaeRope and AlgaeRibbon, specially designed textiles. In a controlled growth experiment, A. esculenta had a similar biomass yield on all materials, but fronds were shorter (23 ± 7%) and thinner on the AlgaeRibbon (42 ± 4%) due to a 3-4-fold higher density of developing sporophytes compared to the twine-longline. In contrast, S. latissima gave a 4-fold higher biomass yield on the AlgaeRibbon in June (4.0 kg m-1), but frond morphology was not different between materials, despite a 4-fold higher sporophyte density on the AlgaeRibbon. The stipe length of both species also increased at the higher sporophyte density on the AlgaeRibbon. The AlgaeRope gave an intermediate response or was similar to the twine-longline. These results show that binder-seeding onto the AlgaeRibbon significantly increases the achieved biomass yield in S. latissima. These results can assist cultivators to select the most appropriate method of kelp cultivation depending on morphological/yield requirements of the end use. Further study is needed on the optimisation of the binder-seeding density and its impact on thallus morphology.

ER-mitochondria contacts promote mitochondrial-derived compartment biogenesis.

Mitochondria are dynamic organelles with essential roles in signaling and metabolism. We recently identified a cellular structure called the mitochondrial-derived compartment (MDC) that is generated from mitochondria in response to amino acid overabundance stress. How cells form MDCs is unclear. Here, we show that MDCs are dynamic structures that form and stably persist at sites of contact between the ER and mitochondria. MDC biogenesis requires the ER-mitochondria encounter structure (ERMES) and the conserved GTPase Gem1, factors previously implicated in lipid exchange and membrane tethering at ER-mitochondria contacts. Interestingly, common genetic suppressors of abnormalities displayed by ERMES mutants exhibit distinct abilities to rescue MDC formation in ERMES-depleted strains and are incapable of rescuing MDC formation in cells lacking Gem1. Thus, the function of ERMES and Gem1 in MDC biogenesis may extend beyond their conventional role in maintaining mitochondrial phospholipid homeostasis. Overall, this study identifies an important function for ER-mitochondria contacts in the biogenesis of MDCs.

Offshore multi-purpose platforms for a Blue Growth: A technological, environmental and socio-economic review.

"Blue Growth" and "Blue Economy" is defined by the World Bank as: "the sustainable use of ocean resources for economic growth, improved livelihoods and jobs, while preserving the health of ocean ecosystem". Multi-purpose platforms (MPPs) can be defined as offshore platforms serving the needs of multiple offshore industries (energy and aquaculture), aim at exploiting the synergies and managing the tensions arising when closely co-locating systems from these industries. Despite a number of previous projects aimed at assessing, from a multidisciplinary point of view, the feasibility of multipurpose platforms, it is here shown that the state-of-the-art has focused mainly on single-purpose devices, and adopting a single discipline (either economic, or social, or technological, or environmental) approach. Therefore, the aim of the present study is to provide a multidisciplinary state of the art review on, whenever possible, multi-purpose platforms, complementing it with single-purpose and/or single discipline literature reviews when not possible. Synoptic tables are provided, giving an overview of the multi-purpose platform concepts investigated, the numerical approaches adopted, and a comprehensive snapshot classifying the references discussed by industry (offshore renewables, aquaculture, both) and by aspect (technological, environmental, socio-economic). The majority of the multi-purpose platform concepts proposed are integrating only multiple offshore renewable energy devices (e.g. hybrid wind-wave), with only few integrating also aquaculture systems. MPPs have significant potential in economizing CAPEX and operational costs for the offshore energy and aquaculture industry by means of concerted spatial planning and sharing of infrastructure.

OPA1 and Angiogenesis: Beyond the Fusion Function.

In this issue of Cell Metabolism, Herkenne et al. (2020) show that the mitochondrial fusion protein OPA1 promotes angiogenesis independent of its function in mitochondrial dynamics, identifying a key new therapeutic target to prevent vascular growth during development and tumor formation.

Lipidomics analysis of juveniles' blue mussels (Mytilus edulis L. 1758), a key economic and ecological species.

Blue mussels (Mytilus edulis L. 1758) are important components of coastal ecosystems and in the economy of rural and coastal areas. The understanding of their physiological processes at key life stages is important both within food production systems and in the management of wild populations. Lipids are crucial molecules for bivalve growth, but their diversity and roles have not been fully characterised. In this study, traditional lipid profiling techniques, such as fatty acid (FA) and lipid class analysis, are combined to untargeted lipidomics to elucidate the lipid metabolism in newly settled spat fed on a range of diets. The evaluated diets included single strains treatments (Cylindrotheca fusiformis CCAP 1017/2 -CYL, Isochrysis galbana CCAP 927/1- ISO, Monodopsis subterranean CCAP 848/1 -MONO, Nannochloropsis oceanica CCAP 849/10- NANNO) and a commercial algae paste (SP). Spat growth was influenced by the diets, which, according to their efficacy were ranked as follows: ISO>NANNO/CYL>SP>MONO. A higher triacylglycerols (TG) content, ranging from 4.23±0.82 µg mgashfree Dry weight (DW)-1 at the beginning of the trial (T0) to 51±15.3 µg mgashfreeDW-1 in ISO, characterised significant growth in the spat, whereas, a reduction of TG (0.3±0.08 µg mgashfreeDW-1 in MONO), mono unsaturated FA-MUFA (from 8.52±1.02 µg mgFAashfreeDW-1 at T0 to 2.81±1.02 µg mgFAashfreeDW-1 in MONO) and polyunsaturated FA-PUFA (from 17.57±2.24 µg mgFAashfreeDW-1 at T0 to 6.19±2.49 µg mgFAashfreeDW-1 in MONO) content characterised poor performing groups. Untargeted lipidomics evidenced how the availability of dietary essential PUFA did not influence only neutral lipids but also the membrane lipids, with changes in lipid molecular species in relation to the essential PUFA provided via the diet. Such changes have the potential to affect spat production cycle and their ability to respond to the surrounding environment. This study evidenced the advantages of coupling different lipid analysis techniques, as each technique disclosed relevant information on nutritional requirements of M. edulis juveniles, expanding the existing knowledge on the physiology of this important species.

Cysteine Toxicity Drives Age-Related Mitochondrial Decline by Altering Iron Homeostasis.

Mitochondria and lysosomes are functionally linked, and their interdependent decline is a hallmark of aging and disease. Despite the long-standing connection between these organelles, the function(s) of lysosomes required to sustain mitochondrial health remains unclear. Here, working in yeast, we show that the lysosome-like vacuole maintains mitochondrial respiration by spatially compartmentalizing amino acids. Defects in vacuole function result in a breakdown in intracellular amino acid homeostasis, which drives age-related mitochondrial decline. Among amino acids, we find that cysteine is most toxic for mitochondria and show that elevated non-vacuolar cysteine impairs mitochondrial respiration by limiting intracellular iron availability through an oxidant-based mechanism. Cysteine depletion or iron supplementation restores mitochondrial health in vacuole-impaired cells and prevents mitochondrial decline during aging. These results demonstrate that cysteine toxicity is a major driver of age-related mitochondrial deterioration and identify vacuolar amino acid compartmentation as a cellular strategy to minimize amino acid toxicity.