Genetic screening reveals phospholipid metabolism as a key regulator of the biosynthesis of the redox-active lipid coenzyme Q.

Mitochondrial energy production and function rely on optimal concentrations of the essential redox-active lipid, coenzyme Q (CoQ). CoQ deficiency results in mitochondrial dysfunction associated with increased mitochondrial oxidative stress and a range of pathologies. What drives CoQ deficiency in many of these pathologies is unknown, just as there currently is no effective therapeutic strategy to overcome CoQ deficiency in humans. To date, large-scale studies aimed at systematically interrogating endogenous systems that control CoQ biosynthesis and their potential utility to treat disease have not been carried out. Therefore, we developed a quantitative high-throughput method to determine CoQ concentrations in yeast cells. Applying this method to the Yeast Deletion Collection as a genome-wide screen, 30 genes not known previously to regulate cellular concentrations of CoQ were discovered. In combination with untargeted lipidomics and metabolomics, phosphatidylethanolamine N-methyltransferase (PEMT) deficiency was confirmed as a positive regulator of CoQ synthesis, the first identified to date. Mechanistically, PEMT deficiency alters mitochondrial concentrations of one-carbon metabolites, characterized by an increase in the S-adenosylmethionine to S-adenosylhomocysteine (SAM-to-SAH) ratio that reflects mitochondrial methylation capacity, drives CoQ synthesis, and is associated with a decrease in mitochondrial oxidative stress. The newly described regulatory pathway appears evolutionary conserved, as ablation of PEMT using antisense oligonucleotides increases mitochondrial CoQ in mouse-derived adipocytes that translates to improved glucose utilization by these cells, and protection of mice from high-fat diet-induced insulin resistance. Our studies reveal a previously unrecognized relationship between two spatially distinct lipid pathways with potential implications for the treatment of CoQ deficiencies, mitochondrial oxidative stress/dysfunction, and associated diseases.

Stress and ageing in yeast.

There has long been speculation about the role of various stresses in ageing. Some stresses have beneficial effects on ageing-dependent on duration and severity of the stress, others have negative effects and the question arises whether these negative effects are causative of ageing or the result of the ageing process. Cellular responses to many stresses are highly coordinated in a concerted way and hence there is a great deal of cross-talk between different stresses. Here the relevant aspects of the coordination of stress responses and the roles of different stresses on yeast cell ageing are discussed, together with the various functions that are involved. The cellular processes that are involved in alleviating the effects of stress on ageing are considered, together with the possible role of early stress events on subsequent ageing of cells.

Ian Dawes-the third Pope-lucky to be a researcher.

Retrospective articles are an excuse for a rosy tinted view of one's life. This fully expurgated version is no exception. No "what the butler saw" or the vilification of enemies that one finds in political autobiographies - merely the account of one born to a generation of those whose forebears never had the chance to go to university and enjoy the subsequent fruits of that education - and of one who by chance stumbled into the world of yeast genetics and molecular biology, who had a lot of fun on the way and who never sought to leave it.

CDP-diacylglycerol synthases regulate the growth of lipid droplets and adipocyte development.

The expansion of lipid droplets (LDs) and the differentiation of preadipocytes are two important aspects of mammalian lipid storage. In this study, we examined the role of CDP-diacylglycerol (DAG) synthases (CDSs), encoded by CDS1 and CDS2 genes in mammals, in lipid storage. CDS enzymes catalyze the formation of CDP-DAG from phosphatidic acid (PA). Knocking down either CDS1 or CDS2 resulted in the formation of giant or supersized LDs in cultured cells. Moreover, depleting CDS1 almost completely blocked the differentiation of 3T3-L1 preadipocytes, whereas depleting CDS2 had a moderate inhibitory effect on adipocyte differentiation. The levels of many PA species were significantly increased upon knocking down CDS1 In contrast, only a small number of PA species were increased upon depleting CDS2 Importantly, the amount of PA in the endoplasmic reticulum was dramatically increased upon knocking down CDS1 or CDS2 Our results suggest that the changes in PA level and localization may underlie the formation of giant LDs as well as the block in adipogenesis in CDS-deficient cells. We have therefore identified CDS1 and CDS2 as important novel regulators of lipid storage, and these results highlight the crucial role of phospholipids in mammalian lipid storage.

Transcriptional and antioxidative responses to endogenous polyunsaturated fatty acid accumulation in yeast.

Pathophysiology of polyunsaturated fatty acids (PUFAs) is associated with aberrant lipid and oxygen metabolism. In particular, under oxidative stress, PUFAs are prone to autocatalytic degradation via peroxidation, leading to formation of reactive aldehydes with numerous potentially harmful effects. However, the pathological and compensatory mechanisms induced by lipid peroxidation are very complex and not sufficiently understood. In our study, we have used yeast capable of endogenous PUFA synthesis in order to understand the effects triggered by PUFA accumulation on cellular physiology of a eukaryotic organism. The mechanisms induced by PUFA accumulation in S. cerevisiae expressing Hevea brasiliensis Δ12-fatty acid desaturase include down-regulation of components of electron transport chain in mitochondria as well as up-regulation of pentose-phosphate pathway and fatty acid ß-oxidation at the transcriptional level. Interestingly, while no changes were observed at the transcriptional level, activities of two important enzymatic antioxidants, catalase and glutathione-S-transferase, were altered in response to PUFA accumulation. Increased intracellular glutathione levels further suggest an endogenous oxidative stress and activation of antioxidative defense mechanisms under conditions of PUFA accumulation. Finally, our data suggest that PUFA in cell membrane causes metabolic changes which in turn lead to adaptation to endogenous oxidative stress.

Actin - a biosensor that determines cell fate in yeasts.

The decision to proliferate, to activate stress response mechanisms or to initiate cell death lies at the heart of the maintenance of a healthy cell population. Within multicellular and colony-forming single-celled organisms, such as yeasts, the functionality of cellular compartments that connect signalling to cell fate must be maintained to maximise adaptability and survival. The actin cytoskeleton is involved in processes such as the regulation of membrane microcompartments, receptor internalisation and the control of master regulatory GTPases, which govern cell decision-making. This affords the actin cytoskeleton a central position within cell response networks. In this sense, a functional actin cytoskeleton is essential to efficiently connect information input to response at the level of the cell. Recent research from fungal, plant and mammalian cells systems has highlighted that actin can trigger apoptotic death in cells that become incompetent to respond to environmental cues. It may also be the case that this property has been appropriated by microorganisms competing for niche environments within a human host. Here, we discuss the research that has been carried out in yeast that links actin to signalling processes and cell fate that supports its role as a biosensor.

Cellular redox homeostasis, reactive oxygen species and replicative ageing in Saccharomyces cerevisiae.

Ageing cells undergo changes in redox homeostasis and acquire high levels of reactive oxygen species (ROS). Because accumulation of ROS involves a change in redox state of cells, functions that are involved in setting redox and maintaining redox homeostasis are very relevant to an understanding of the possible roles of redox homeostasis and ROS in ageing. This review discusses these aspects of ROS in relation to replicative ageing in the model organism Saccharomyces cerevisiae, with reference to ROS generated in cells; cellular responses to oxidative stress; and how cells maintain redox homeostasis in different cellular compartments. It also considers when ROS generation begins as cells age, which ROS species are relevant to ageing and which cellular compartments and processes may contribute ROS to the ageing process. The discussion also covers the heterogeneity of cells with respect to ROS accumulation at particular cell ages, and the possibility of testing the oxidative theory of ageing in yeast cells.

Saccharomyces cerevisiae genes involved in survival of heat shock.

The heat-shock response in cells, involving increased transcription of a specific set of genes in response to a sudden increase in temperature, is a highly conserved biological response occurring in all organisms. Despite considerable attention to the processes activated during heat shock, less is known about the role of genes in survival of a sudden temperature increase. Saccharomyces cerevisiae genes involved in the maintenance of heat-shock resistance in exponential and stationary phase were identified by screening the homozygous diploid deletants in nonessential genes and the heterozygous diploid mutants in essential genes for survival after a sudden shift in temperature from 30 to 50°. More than a thousand genes were identified that led to altered sensitivity to heat shock, with little overlap between them and those previously identified to affect thermotolerance. There was also little overlap with genes that are activated or repressed during heat-shock, with only 5% of them regulated by the heat-shock transcription factor. The target of rapamycin and protein kinase A pathways, lipid metabolism, vacuolar H(+)-ATPase, vacuolar protein sorting, and mitochondrial genome maintenance/translation were critical to maintenance of resistance. Mutants affected in l-tryptophan metabolism were heat-shock resistant in both growth phases; those affected in cytoplasmic ribosome biogenesis and DNA double-strand break repair were resistant in stationary phase, and in mRNA catabolic processes in exponential phase. Mutations affecting mitochondrial genome maintenance were highly represented in sensitive mutants. The cell division transcription factor Swi6p and Hac1p involved in the unfolded protein response also play roles in maintenance of heat-shock resistance.

Maintenance of mitochondrial morphology by autophagy and its role in high glucose effects on chronological lifespan of Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, mitochondrial morphology changes when cells are shifted between nonfermentative and fermentative carbon sources. Here, we show that cells of S. cerevisiae grown in different glucose concentrations display different mitochondrial morphologies. The morphology of mitochondria in the cells growing in 0.5% glucose was similar to that of mitochondria in respiring cells. However, the mitochondria of cells growing in higher glucose concentrations (2% and 4%) became fragmented after growth in these media, due to the production of acetic acid; however, the fragmentation was not due to intracellular acidification. From a screen of mutants involved in sensing and utilizing nutrients, cells lacking TOR1 had reduced mitochondrial fragmentation, and autophagy was found to be essential for this reduction. Mitochondrial fragmentation in cells grown in high glucose was reversible by transferring them into conditioned medium from a culture grown on 0.5% glucose. Similarly, the chronological lifespan of cells grown in high glucose medium was reduced, and this phenotype could be reversed when cells were transferred to low glucose conditioned medium. These data indicate that chronological lifespan seems correlated with mitochondrial morphology of yeast cells and that both phenotypes can be influenced by factors from conditioned medium of cultures grown in low glucose medium.

Superoxide radicals have a protective role during H2O2 stress.

Reactive oxygen species (ROS) consist of potentially toxic, partly reduced oxygen species and free radicals. After H(2)O(2) treatment, yeast cells significantly increase superoxide radical production. Respiratory chain complex III and possibly cytochrome b function are essential for this increase. Disruption of complex III renders cells sensitive to H(2)O(2) but not to the superoxide radical generator menadione. Of interest, the same H(2)O(2)-sensitive mutant strains have the lowest superoxide radical levels, and strains with the highest resistance to H(2)O(2) have the highest levels of superoxide radicals. Consistent with this correlation, overexpression of superoxide dismutase increases sensitivity to H(2)O(2), and this phenotype is partially rescued by addition of small concentrations of menadione. Small increases in levels of mitochondrially produced superoxide radicals have a protective effect during H(2)O(2)-induced stress, and in response to H(2)O(2), the wild-type strain increases superoxide radical production to activate this defense mechanism. This provides a direct link between complex III as the main source of ROS and its role in defense against ROS. High levels of the superoxide radical are still toxic. These opposing, concentration-dependent roles of the superoxide radical comprise a form of hormesis and show one ROS having a hormetic effect on the toxicity of another.

Cellular responses to L-serine in Saccharomyces cerevisiae: roles of general amino acid control, compartmentalization, and aspartate synthesis.

In addition to its other roles, L-serine functions in one-carbon metabolism and is interconvertable with glycine via serine hydroxymethyltransferases. However, the transcriptional response by Saccharomyces cerevisiae to L-serine addition is markedly different from that to glycine, with L-serine acting as a nutrient source rather than one-carbon units. Following addition of excess L-serine, 743 genes showed significant expression changes. Induced functions included amino acid synthesis, some stress responses, and FeS metabolism, while ribosomal RNA processing, ribosome biogenesis and hexose transport were repressed. A co-regulated network of ten transcription factors could together control more than 90% of the induced and repressed genes forming a general response to changes induced by other amino acids or stresses and including the general amino acid control system usually activated in response to starvation for amino acids. A specific response to L-serine was induction of CHA1 encoding serine (threonine) dehydratase. L-serine addition resulted in a substantial transient increase in L-aspartate, which is, rather than L-glutamate, the major metabolite for short-term storage of ammonia derived from degradation of L-serine. L-aspartate synthesis was exclusively through mitochondrial metabolism of L-serine to pyruvate and ammonia, involving Cha1p, cytoplasmic pyruvate carboxylases Pyc1p and Pyc2p, and the cytoplasmic aspartate aminotransferase Aat2p.

Distinct redox regulation in sub-cellular compartments in response to various stress conditions in Saccharomyces cerevisiae.

Responses to many growth and stress conditions are assumed to act via changes to the cellular redox status. However, direct measurement of pH-adjusted redox state during growth and stress has never been carried out. Organellar redox state (E GSH) was measured using the fluorescent probes roGFP2 and pHluorin in Saccharomyces cerevisiae. In particular, we investigated changes in organellar redox state in response to various growth and stress conditions to better understand the relationship between redox-, oxidative- and environmental stress response systems. E GSH values of the cytosol, mitochondrial matrix and peroxisome were determined in exponential and stationary phase in various media. These values (-340 to -350 mV) were more reducing than previously reported. Interestingly, sub-cellular redox state remained unchanged when cells were challenged with stresses previously reported to affect redox homeostasis. Only hydrogen peroxide and heat stress significantly altered organellar redox state. Hydrogen peroxide stress altered the redox state of the glutathione disulfide/glutathione couple (GSSG, 2H(+)/2GSH) and pH. Recovery from moderate hydrogen peroxide stress was most rapid in the cytosol, followed by the mitochondrial matrix, with the peroxisome the least able to recover. Conversely, the bulk of the redox shift observed during heat stress resulted from alterations in pH and not the GSSG, 2H(+)/2GSH couple. This study presents the first direct measurement of pH-adjusted redox state in sub-cellular compartments during growth and stress conditions. Redox state is distinctly regulated in organelles and data presented challenge the notion that perturbation of redox state is central in the response to many stress conditions.

Oxidative stress and neurodegeneration: the yeast model system.

In this chapter we are treating yeast cells as a model for oxidative stress response and the consequences of oxidative stress which are one cause for a number of human diseases, including neurodegenerative diseases, which form the main part of this paper. All such model building depends on orthologous relations between highly conserved yeast and human genes, which are easily recognized by sequence comparisons, but much more difficult to prove functionally. Previously we have treated Friedreich's ataxia, while presently we are describing in detail the neuronal ceroid lipofuscinoses, among them Batten disease. A general overview is given how yeast can aid current research in three of the most devastating and at the same time quantitatively most important neurodegenerative diseases of old age: Alzheimer's, Huntington's, and Parkinson's disease. In the ensuing part of the chapter, we describe yeast as model for metabolic regulation and hence as a model for inborn errors of metabolism that are in some instances very faithfully mirrored by introducing the same point mutations into yeast cells which are known from patients.

The AAA ATPase VPS4/SKD1 regulates endosomal cholesterol trafficking independently of ESCRT-III.

The exit of low-density lipoprotein derived cholesterol (LDL-C) from late endosomes (LE)/lysosomes (Ly) is mediated by Niemann-Pick C1 (NPC1), a multipass integral membrane protein on the limiting membranes of LE/Ly, and by NPC2, a cholesterol-binding protein in the lumen of LE/Ly. NPC2 delivers cholesterol to the N-terminal domain of NPC1, which is believed to insert cholesterol into the limiting membrane for subsequent transport to other subcellular organelles. Few cytoplasmic factors have been identified to govern cholesterol efflux from LE/Ly, and much less is known about the underlying molecular mechanisms. Here we establish VPS4, an AAA ATPase that has a well-established role in disassembling the ESCRT (endosomal sorting complex required for transport)-III polymer, as an important regulator of endosomal cholesterol transport. Knocking down VPS4 in HeLa cells resulted in prominent accumulation of LDL-C in LE/Ly, and disrupted cholesterol homeostatic responses at the endoplasmic reticulum. The level and localization of NPC1 and NPC2 appeared to be normal in VPS4 knockdown cells. Importantly, depleting any of the ESCRT-III components did not exert a significant effect on endosomal cholesterol transport. Our results thus identify an important cytoplasmic regulator of endosomal cholesterol trafficking and represent the first functional separation of VPS4 from ESCRT-III.

Redox control of cell proliferation.

Cell proliferation is regulated by multiple signaling pathways and stress surveillance systems to ensure cell division takes place with fidelity. In response to oxidative stress, cells arrest in the cell-cycle and aberrant redox control of proliferation underlies the pathogenesis of many diseases including cancer and neurodegenerative disorders. Redox sensing of cell-cycle regulation has recently been shown to involve reactive cysteine thiols that function as redox sensors in cell-cycle regulators. By modulating cell-cycle regulators these redox-active thiols ensure cell division is executed at the right redox environment. This review summarizes recent findings on regulation of cell division by the oxidation of cysteines in cell division regulators and the potential of targeting these critical cysteine residues for cancer therapy.

Accumulation of squalene is associated with the clustering of lipid droplets.

The isoprenoid squalene is an important precursor for the biosynthesis of sterols. The cellular storage of squalene and its impact on membrane properties have been the subject of recent investigations. In a screen for abnormal lipid droplet morphology and distribution in the yeast Saccharomyces cerevisiae, we found significant lipid droplet clustering (arbitrarily defined as an aggregation of six or more lipid droplets) in a number of mutants (e.g. erg1) that are defective in sterol biosynthesis. Interestingly, these mutants are also characterized by accumulation of large amounts of squalene. Reducing the level of squalene in these mutants restored normal lipid droplet distribution. Moreover, inhibition of squalene monooxygenase in two mammalian cell lines (CHO-K1 and 3T3-L1) by terbinafine also resulted in lipid droplet clustering. These results indicate that the level of squalene may affect the growth and distribution of lipid droplets.

A genome-wide screen in yeast identifies specific oxidative stress genes required for the maintenance of sub-cellular redox homeostasis.

Maintenance of an optimal redox environment is critical for appropriate functioning of cellular processes and cell survival. Despite the importance of maintaining redox homeostasis, it is not clear how the optimal redox potential is sensed and set, and the processes that impact redox on a cellular/organellar level are poorly understood. The genetic bases of cellular redox homeostasis were investigated using a green fluorescent protein (GFP) based redox probe, roGFP2 and a pH sensitive GFP-based probe, pHluorin. The use of roGFP2, in conjunction with pHluorin, enabled determination of pH-adjusted sub-cellular redox potential in a non-invasive and real-time manner. A genome-wide screen using both the non-essential and essential gene collections was carried out in Saccharomyces cerevisiae using cytosolic-roGFP2 to identify factors essential for maintenance of cytosolic redox state under steady-state conditions. 102 genes of diverse function were identified that are required for maintenance of cytosolic redox state. Mutations in these genes led to shifts in the half-cell glutathione redox potential by 75-10 mV. Interestingly, some specific oxidative stress-response processes were identified as over-represented in the data set. Further investigation of the role of oxidative stress-responsive systems in sub-cellular redox homeostasis was conducted using roGFP2 constructs targeted to the mitochondrial matrix and peroxisome and E(GSH) was measured in cells in exponential and stationary phase. Analyses allowed for the identification of key redox systems on a sub-cellular level and the identification of novel genes involved in the regulation of cellular redox homeostasis.

The endosomal protein-sorting receptor sortilin has a role in trafficking α-1 antitrypsin.

Up to 1 in 3000 individuals in the United States have α-1 antitrypsin deficiency, and the most common cause of this disease is homozygosity for the antitrypsin-Z variant (ATZ). ATZ is inefficiently secreted, resulting in protein deficiency in the lungs and toxic polymer accumulation in the liver. However, only a subset of patients suffer from liver disease, suggesting that genetic factors predispose individuals to liver disease. To identify candidate factors, we developed a yeast ATZ expression system that recapitulates key features of the disease-causing protein. We then adapted this system to screen the yeast deletion mutant collection to identify conserved genes that affect ATZ secretion and thus may modify the risk for developing liver disease. The results of the screen and associated assays indicate that ATZ is degraded in the vacuole after being routed from the Golgi. In fact, one of the strongest hits from our screen was Vps10, which can serve as a receptor for the delivery of aberrant proteins to the vacuole. Because genome-wide association studies implicate the human Vps10 homolog, sortilin, in cardiovascular disease, and because hepatic cell lines that stably express wild-type or mutant sortilin were recently established, we examined whether ATZ levels and secretion are affected by sortilin. As hypothesized, sortilin function impacts the levels of secreted ATZ in mammalian cells. This study represents the first genome-wide screen for factors that modulate ATZ secretion and has led to the identification of a gene that may modify disease severity or presentation in individuals with ATZ-associated liver disease.

Oxidative stresses and ageing.

Oxidative damage to cellular constituents has frequently been associated with aging in a wide range of organisms. The power of yeast genetics and biochemistry has provided the opportunity to analyse in some detail how reactive oxygen and nitrogen species arise in cells, how cells respond to the damage that these reactive species cause, and to begin to dissect how these species may be involved in the ageing process. This chapter reviews the major sources of reactive oxygen species that occur in yeast cells, the damage they cause and how cells sense and respond to this damage.

The role of mitochondria in the aging processes of yeast.

This chapter reviews the role of mitochondria and of mitochondrial metabolism in the aging processes of yeast and the existing evidence for the "mitochondrial theory of aging mitochondrial theory of aging ". Mitochondria are the major source of ATP in the eukaryotic cell but are also a major source of reactive oxygen species reactive oxygen species (ROS) and play an important role in the process of apoptosis and aging. We are discussing the mitochondrial theory of aging mitochondrial theory of aging (TOA), its origin, similarity with other TOAs, and its ramifications which developed in recent decades. The emphasis is on mother cell-specific aging mother cell-specific aging and the RLS (replicative lifespan) with only a short treatment of CLS (chronological lifespan). Both of these aging processes may be relevant to understand also the aging of higher organisms, but they are biochemically very different, as shown by the fact the replicative aging occurs on rich media and is a defect in the replicative capacity of mother cells, while chronological aging occurs in postmitotic cells that are under starvation conditions in stationary phase leading to loss of viability, as discussed elsewhere in this book. In so doing we also give an overview of the similarities and dissimilarities of the various aging processes of the most often used model organisms for aging research with respect to the mitochondrial theory of aging mitochondrial theory of aging.