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.

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).

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.

An early age increase in vacuolar pH limits mitochondrial function and lifespan in yeast.

Mitochondria have a central role in ageing. They are considered to be both a target of the ageing process and a contributor to it. Alterations in mitochondrial structure and function are evident during ageing in most eukaryotes, but how this occurs is poorly understood. Here we identify a functional link between the lysosome-like vacuole and mitochondria in Saccharomyces cerevisiae, and show that mitochondrial dysfunction in replicatively aged yeast arises from altered vacuolar pH. We found that vacuolar acidity declines during the early asymmetric divisions of a mother cell, and that preventing this decline suppresses mitochondrial dysfunction and extends lifespan. Surprisingly, changes in vacuolar pH do not limit mitochondrial function by disrupting vacuolar protein degradation, but rather by reducing pH-dependent amino acid storage in the vacuolar lumen. We also found that calorie restriction promotes lifespan extension at least in part by increasing vacuolar acidity via conserved nutrient-sensing pathways. Interestingly, although vacuolar acidity is reduced in aged mother cells, acidic vacuoles are regenerated in newborn daughters, coinciding with daughter cells having a renewed lifespan potential. Overall, our results identify vacuolar pH as a critical regulator of ageing and mitochondrial function, and outline a potentially conserved mechanism by which calorie restriction delays the ageing process. Because the functions of the vacuole are highly conserved throughout evolution, we propose that lysosomal pH may modulate mitochondrial function and lifespan in other eukaryotic cells.

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.

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.

Dap1/PGRMC1 binds and regulates cytochrome P450 enzymes.

Cytochrome P450 enzymes are heme-dependent monoxygenases that play a central role in human physiology. Despite the numerous physiological processes that P450 enzymes impact, the electron donors P450 oxidoreductase and cytochrome b5 are the only proteins known to interact with and modulate the activity of ER microsomal P450s. Here, we report that Dap1/PGRMC1 is required for ER P450 function in yeast and humans. We show that S. pombe Dap1 is a hemoprotein that binds and positively regulates Cyp51A1 and Cyp61A1, two P450s required for sterol biosynthesis. Similarly, loss of human PGRMC1 reduces activity of Cyp51A1, blocking cholesterol synthesis and increasing production of toxic sterol intermediates. PGRMC1 stably binds Cyp51A1 and human P450s from three additional families including Cyp3A4, which metabolizes pharmaceutical compounds. These findings demonstrate that PGRMC1 is required for P450 activity and suggest that interindividual variation in PGRMC1 function may impact multiple biochemical pathways and drug metabolism.

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.

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.

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.

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.

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.

Sterol regulatory element binding protein is a principal regulator of anaerobic gene expression in fission yeast.

Fission yeast sterol regulatory element binding protein (SREBP), called Sre1p, functions in an oxygen-sensing pathway to allow adaptation to fluctuating oxygen concentrations. The Sre1p-Scp1p complex responds to oxygen-dependent sterol synthesis as an indirect measure of oxygen availability. To examine the role of Sre1p in anaerobic gene expression in Schizosaccharomyces pombe, we performed transcriptional profiling experiments after a shift to anaerobic conditions for 1.5 h. Of the 4,940 genes analyzed, expression levels of 521 (10.5%) and 686 (13.9%) genes were significantly increased and decreased, respectively, under anaerobic conditions. Sre1p controlled 68% of genes induced > or = 2-fold. Oxygen-requiring biosynthetic pathways for ergosterol, heme, sphingolipid, and ubiquinone were primary targets of Sre1p. Induction of glycolytic genes and repression of mitochondrial oxidative phosphorylation genes largely did not require Sre1p. Using chromatin immunoprecipitation, we demonstrated that Sre1p acts directly at target gene promoters and stimulates its own transcription under anaerobic conditions. sre1+ promoter analysis identified two DNA elements that are both necessary and sufficient for oxygen-dependent, Sre1p-dependent transcription. Interestingly, these elements are homologous to sterol regulatory elements bound by mammalian SREBP, highlighting the evolutionary conservation between Sre1p and SREBP. We conclude that Sre1p is a principal activator of anaerobic gene expression, upregulating genes required for nonrespiratory oxygen consumption.

Knowing When to Let Go: Lysosomes Regulate Inter-Mitochondrial Tethering.

In this issue of Developmental Cell, Wong et al. (2019) show that the lysosomal GTPase Rab7 regulates inter-mitochondrial contacts to control mitochondrial motility and identify dysregulated inter-mitochondrial tethering as a common theme in Charcot-Marie-Tooth (CMT) type 2 disease.

Rsp5 and Mdm30 reshape the mitochondrial network in response to age-induced vacuole stress.

Mitochondrial decline is a hallmark of aging, and cells are equipped with many systems to regulate mitochondrial structure and function in response to stress and metabolic alterations. Here, using budding yeast, we identify a proteolytic pathway that contributes to alterations in mitochondrial structure in aged cells through control of the mitochondrial fusion GTPase Fzo1. We show that mitochondrial fragmentation in old cells correlates with reduced abundance of Fzo1, which is triggered by functional alterations in the vacuole, a known early event in aging. Fzo1 degradation is mediated by a proteolytic cascade consisting of the E3 ubiquitin ligases SCFMdm30 and Rsp5, and the Cdc48 cofactor Doa1. Fzo1 proteolysis is activated by metabolic stress that arises from vacuole impairment, and loss of Fzo1 degradation severely impairs mitochondrial structure and function. Together, these studies identify a new mechanism for stress-responsive regulation of mitochondrial structure that is activated during cellular aging.

Advancing the aging biology toolkit.

A new device for isolating large quantities of old yeast cells expands the experimental boundaries of aging research.

Systematic mapping of contact sites reveals tethers and a function for the peroxisome-mitochondria contact.

The understanding that organelles are not floating in the cytosol, but rather held in an organized yet dynamic interplay through membrane contact sites, is altering the way we grasp cell biological phenomena. However, we still have not identified the entire repertoire of contact sites, their tethering molecules and functions. To systematically characterize contact sites and their tethering molecules here we employ a proximity detection method based on split fluorophores and discover four potential new yeast contact sites. We then focus on a little-studied yet highly disease-relevant contact, the Peroxisome-Mitochondria (PerMit) proximity, and uncover and characterize two tether proteins: Fzo1 and Pex34. We genetically expand the PerMit contact site and demonstrate a physiological function in ß-oxidation of fatty acids. Our work showcases how systematic analysis of contact site machinery and functions can deepen our understanding of these structures in health and disease.

The crucial impact of lysosomes in aging and longevity.

Lysosomes are the main catabolic organelles of a cell and play a pivotal role in a plethora of cellular processes, including responses to nutrient availability and composition, stress resistance, programmed cell death, plasma membrane repair, development, and cell differentiation. In line with this pleiotropic importance for cellular and organismal life and death, lysosomal dysfunction is associated with many age-related pathologies like Parkinson's and Alzheimer's disease, as well as with a decline in lifespan. Conversely, targeting lysosomal functional capacity is emerging as a means to promote longevity. Here, we analyze the current knowledge on the prominent influence of lysosomes on aging-related processes, such as their executory and regulatory roles during general and selective macroautophagy, or their storage capacity for amino acids and ions. In addition, we review and discuss the roles of lysosomes as active players in the mechanisms underlying known lifespan-extending interventions like, for example, spermidine or rapamycin administration. In conclusion, this review aims at critically examining the nature and pliability of the different layers, in which lysosomes are involved as a control hub for aging and longevity.

Selective sorting and destruction of mitochondrial membrane proteins in aged yeast.

Mitochondrial dysfunction is a hallmark of aging, and underlies the development of many diseases. Cells maintain mitochondrial homeostasis through a number of pathways that remodel the mitochondrial proteome or alter mitochondrial content during times of stress or metabolic adaptation. Here, using yeast as a model system, we identify a new mitochondrial degradation system that remodels the mitochondrial proteome of aged cells. Unlike many common mitochondrial degradation pathways, this system selectively removes a subset of membrane proteins from the mitochondrial inner and outer membranes, while leaving the remainder of the organelle intact. Selective removal of preexisting proteins is achieved by sorting into a mitochondrial-derived compartment, or MDC, followed by release through mitochondrial fission and elimination by autophagy. Formation of MDCs requires the import receptors Tom70/71, and failure to form these structures exacerbates preexisting mitochondrial dysfunction, suggesting that the MDC pathway provides protection to mitochondria in times of stress.

Power(2): the power of yeast genetics applied to the powerhouse of the cell.

The budding yeast Saccharomyces cerevisiae has served as a remarkable model organism for numerous seminal discoveries in biology. This paradigm extends to the mitochondria, a central hub for cellular metabolism, where studies in yeast have helped to reinvigorate the field and launch an exciting new era in mitochondrial biology. Here we discuss a few recent examples in which yeast research has laid a foundation for our understanding of evolutionarily conserved mitochondrial processes and functions, from key factors and pathways involved in the assembly of oxidative phosphorylation (OXPHOS) complexes to metabolite transport, lipid metabolism, and interorganelle communication. We also highlight new areas of yeast mitochondrial biology that are likely to aid in our understanding of the mitochondrial etiology of disease in the future.