Starvation Induced Cell Death in Autophagy-Defective Yeast Mutants Is Caused by Mitochondria Dysfunction

Autophagy is a highly-conserved cellular degradation and recycling system that is essential for cell survival during nutrient starvation. The loss of viability had been used as an initial screen to identify autophagy-defective (atg) mutants of the yeast Saccharomyces cerevisiae, but the mechanism of cell death in these mutants has remained unclear. When cells grown in a rich medium were transferred to a synthetic nitrogen starvation media, secreted metabolites lowered the extracellular pH below 3.0 and autophagy-defective mutants mostly died. We found that buffering of the starvation medium dramatically restored the viability of atg mutants. In response to starvation, wild-type (WT) cells were able to upregulate components of the respiratory pathway and ROS (reactive oxygen species) scavenging enzymes, but atg mutants lacked this synthetic capacity. Consequently, autophagy-defective mutants accumulated the high level of ROS, leading to deficient respiratory function, resulting in the loss of mitochondria DNA (mtDNA). We also showed that mtDNA deficient cells are subject to cell death under low pH starvation conditions. Taken together, under starvation conditions non-selective autophagy, rather than mitophagy, plays an essential role in preventing ROS accumulation, and thus in maintaining mitochondria function. The failure of response to starvation is the major cause of cell death in atg mutants

Interfering with Glycolysis Causes Sir2-Dependent Hyper-Recombination of Saccharomyces cerevisiae Plasmids

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key metabolic regulator implicated in a variety of cellular processes. It functions as a glycolytic enzyme, a protein kinase, and a metabolic switch under oxidative stress. Its enzymatic inactivation causes a major shift in the primary carbohydrate flux. Furthermore, the protein is implicated in regulating transcription, ER-to-Golgi transport, and apoptosis. We found that Saccharomyces cerevisiae cells null for all GAPDH paralogues (Tdh1, Tdh2, and Tdh3) survived the counter-selection of a GAPDH–encoding plasmid when the NAD+ metabolizing deacetylase Sir2 was overexpressed. This phenotype required a fully functional copy of SIR2 and resulted from hyper-recombination between S. cerevisiae plasmids. In the wild-type background, GAPDH overexpression increased the plasmid recombination rate in a growth-condition dependent manner. We conclude that GAPDH influences yeast episome stability via Sir2 and propose a model for the interplay of Sir2, GAPDH, and the glycolytic flux

A Microarray-Based Genetic Screen for Yeast Chronological Aging Factors

Model organisms have played an important role in the elucidation of multiple genes and cellular processes that regulate aging. In this study we utilized the budding yeast, Saccharomyces cerevisiae, in a large-scale screen for genes that function in the regulation of chronological lifespan, which is defined by the number of days that non-dividing cells remain viable. A pooled collection of viable haploid gene deletion mutants, each tagged with unique identifying DNA “bar-code” sequences was chronologically aged in liquid culture. Viable mutants in the aging population were selected at several time points and then detected using a microarray DNA hybridization technique that quantifies abundance of the barcode tags. Multiple short- and long-lived mutants were identified using this approach. Among the confirmed short-lived mutants were those defective for autophagy, indicating a key requirement for the recycling of cellular organelles in longevity. Defects in autophagy also prevented lifespan extension induced by limitation of amino acids in the growth media. Among the confirmed long-lived mutants were those defective in the highly conserved de novo purine biosynthesis pathway (the ADE genes), which ultimately produces IMP and AMP. Blocking this pathway extended lifespan to the same degree as calorie (glucose) restriction. A recently discovered cell-extrinsic mechanism of chronological aging involving acetic acid secretion and toxicity was suppressed in a long-lived ade4Δ mutant and exacerbated by a short-lived atg16Δ autophagy mutant. The identification of multiple novel effectors of yeast chronological lifespan will greatly aid in the elucidation of mechanisms that cells and organisms utilize in slowing down the aging process

Natural Polymorphism in BUL2 Links Cellular Amino Acid Availability with Chronological Aging and Telomere Maintenance in Yeast

Aging and longevity are considered to be highly complex genetic traits. In order to gain insight into aging as a polygenic trait, we employed an outbred Saccharomyces cerevisiae model, generated by crossing a vineyard strain RM11 and a laboratory strain S288c, to identify quantitative trait loci that control chronological lifespan. Among the major loci that regulate chronological lifespan in this cross, one genetic linkage was found to be congruent with a previously mapped locus that controls telomere length variation. We found that a single nucleotide polymorphism in BUL2, encoding a component of an ubiquitin ligase complex involved in trafficking of amino acid permeases, controls chronological lifespan and telomere length as well as amino acid uptake. Cellular amino acid availability changes conferred by the BUL2 polymorphism alter telomere length by modulating activity of a transcription factor Gln3. Among the GLN3 transcriptional targets relevant to this phenotype, we identified Wtm1, whose upregulation promotes nuclear retention of ribonucleotide reductase (RNR) components and inhibits the assembly of the RNR enzyme complex during S-phase. Inhibition of RNR is one of the mechanisms by which Gln3 modulates telomere length. Identification of a polymorphism in BUL2 in this outbred yeast population revealed a link among cellular amino acid availability, chronological lifespan, and telomere length control

Parallel Profiling of Fission Yeast Deletion Mutants for Proliferation and for Lifespan During Long-Term Quiescence

Genetic factors underlying aging are remarkably conserved from yeast to human. The fission yeast Schizosaccharomyces pombe is an emerging genetic model to analyze cellular aging. Chronological lifespan (CLS) has been studied in stationary-phase yeast cells depleted for glucose, which only survive for a few days. Here, we analyzed CLS in quiescent S. pombe cells deprived of nitrogen, which arrest in a differentiated, G0-like state and survive for more than 2 months. We applied parallel mutant phenotyping by barcode sequencing (Bar-seq) to assay pooled haploid deletion mutants as they aged together during longterm quiescence. As expected, mutants with defects in autophagy or quiescence were under-represented or not detected. Lifespan scores could be calculated for 1199 mutants. We focus the discussion on the 48 most long-lived mutants, including both known aging genes in other model systems and genes not previously implicated in aging. Genes encoding membrane proteins were particularly prominent as pro-aging factors. We independently verified the extended CLS in individual assays for 30 selected mutants, showing the efficacy of the screen. We also applied Bar-seq to profile all pooled deletion mutants for proliferation under a standard growth condition. Unlike for stationary-phase cells, no inverse correlation between growth and CLS of quiescent cells was evident. These screens provide a rich resource for further studies, and they suggest that the quiescence model can provide unique, complementary insights into cellular aging

Dynamic mechanical properties of artificially aged double base rocket propellant and the possibilities for the prediction of their service lifetime

The ageing of double base (DB) rocket propellants, as a consequence of the chemical reactions and physical processes that take place over time, has a significant effect on their relevant properties, such as chemical composition and mechanical and ballistic properties. The changes to relevant properties limit the safe and reliable service life of DB rocket propellants. Accordingly, numerous research efforts have been undertaken to find reliable methods to measure the changes caused by ageing in order to assess the quality of DB rocket propellants at a given moment of their lifetime, and to predict their remaining service lifetime. In this work we studied the dynamic mechanical properties of DB rocket propellant artificially aged at temperatures of 80, 85 and 90 °C, in order to detect and quantify changes in the dynamic mechanical properties caused by ageing, and to investigate the possibilities for the prediction of service lifetime. Dynamic mechanical properties were studied using a dynamic mechanical analyser (DMA). The results obtained have shown that ageing causes significant changes in the storage modulus (E´), the loss modulus (E˝) and the tan δ curves’ shape and position. These changes are quantified by following some characteristic points on the E´-T, E˝-T, and tan δ-T curves (e.g. glass transition temperatures; storage modulus, loss modulus and tan δ at characteristic temperatures, etc.). It has been found that the monitored parameters are temperature and time dependent, and that they can be shown to be functions of the so called ‘reduced time of artificial ageing’. In addition, it has been found that, on the basis of known changes in viscoelastic properties as a function of time and ageing temperature, and the known kinetic parameters of the ageing process, it is possible to calculate (determine) the change in the properties at any ageing temperature provided that the mechanism of the ageing process does not change. Unfortunately, the use of kinetic parameters obtained by artificial ageing at high temperatures (above 60 °C) for the prediction of the propellant lifetime will not give reliable results, because the mechanisms of ageing at 85 °C and 25 °C are not the same.Published versio

The applicability of chromatographic methods in the investigation of ageing processes in double base rocket propellants

The ageing of double base (DB) rocket propellants is the result of chemical decomposition reactions and physical processes, causing degradation of a number of relevant propellant properties (such as reduction in stabilizer and nitroglycerine (NG) content, reduction of the mean molecular mass of nitrocellulose (NC) etc.), which is reflected in a decrease in the reliable service life time of DB propellants. This is the reason why the study of processes of ageing and their consequences (effects) is so important. In this paper we have studied the kinetics of DB rocket propellant decomposition during their artificial ageing, i.e. at elevated temperatures. The kinetic parameters were obtained by measurements of the stabilizer/Ethyl Centralite (EC) content and the mean molecular mass reduction of NC, during artificial ageing at temperatures of 80, 85 and 90 °C. Consumption of the EC was observed using High Performance Liquid Chromatography (HPLC), whilst the reduction in the mean molecular mass of NC was monitored using Gel Permeation Chromatography (GPC). It has been shown that artificial ageing of DB propellant causes significant EC consumption and a reduction in the mean molecular mass of NC, from the very beginning of ageing. EC is entirely consumed after 120 days at 80 °C, and is followed by the intensive reactions of NC decomposition. Significant changes in the mean molecular mass of NC starts after 60 days of ageing at 90 °C (or ~250 days at 80 °C). The results obtained from the kinetic data have shown that the activation energy of DB propellant decomposition, determined on the basis of changes in the mean molecular mass of NC is 145.09 kJ·mol-1, whilst the activation energyof decomposition obtained on the basis of EC consumption is 142.98 kJ·mol-1, which is consistent with available literature values [1, 2].Published versio