Stationary Phase Yeast Research Articles

Metabolic deregulation associated with aging modulates protein aggregation in the yeast model of Huntington’s disease

Huntington’s disease is associated with increased CAG repeat resulting in an expanded polyglutamine tract in the protein Huntingtin (HTT) leading to its aggregation resulting in neurodegeneration. Previous studies have shown that N-terminal HTT with 46Q aggregated in the stationary phase but not the logarithmic phase in the yeast model of HD. We carried out a metabolomic analysis of logarithmic and stationary phase yeast model of HD expressing different polyQ lengths attached to N-terminal HTT tagged with enhanced green fluorescent protein (EGFP). The results show significant changes in the metabolic profile and deregulated pathways in stationary phase cells compared to logarithmic phase cells. Comparison of metabolic pathways obtained from logarithmic phase 46Q versus 25Q with those obtained for presymptomatic HD patients from our previous study and drosophila model of HD showed considerable overlap. The arginine biosynthesis pathway emerged as one of the key pathways that is common in stationary phase yeast compared to logarithmic phase and HD patients. Treatment of yeast with arginine led to a significant decrease, while transfer to arginine drop-out media led to a significant increase in the size of protein aggregates in both logarithmic and stationary phase yeast model of HD. Knockout of arginine transporters in the endoplasmic reticulum and vacuole led to a significant decrease in mutant HTT aggregation. Overall our results highlight arginine as a critical metabolite that modulates the aggregation of mutant HTT and disease progression in HD. Communicated by Ramaswamy H. Sarma

Fatty Acyl Coenzyme A Synthetase Fat1p Regulates Vacuolar Structure and Stationary-Phase Lipophagy in Saccharomyces cerevisiae.

During yeast stationary phase, a single spherical vacuole (lysosome) is created by the fusion of several small ones. Moreover, the vacuolar membrane is reconstructed into two distinct microdomains. Little is known, however, about how cells maintain vacuolar shape or regulate their microdomains. Here, we show that Fat1p, a fatty acyl coenzyme A (acyl-CoA) synthetase and fatty acid transporter, and not the synthetases Faa1p and Faa4p, is essential for vacuolar shape preservation, the development of vacuolar microdomains, and cell survival in stationary phase of the yeast Saccharomyces cerevisiae. Furthermore, Fat1p negatively regulates general autophagy in both log- and stationary-phase cells. In contrast, Fat1p promotes lipophagy, as the absence of FAT1 limits the entry of lipid droplets into the vacuole and reduces the degradation of liquid droplet (LD) surface proteins. Notably, supplementing with unsaturated fatty acids or overexpressing the desaturase Ole1p can reverse all aberrant phenotypes caused by FAT1 deficiency. We propose that Fat1p regulates stationary phase vacuolar morphology, microdomain differentiation, general autophagy, and lipophagy by controlling the degree of fatty acid saturation in membrane lipids. IMPORTANCE The ability to sense environmental changes and adjust the levels of cellular metabolism is critical for cell viability. Autophagy is a recycling process that makes the most of already-existing energy resources, and the vacuole/lysosome is the ultimate autophagic processing site in cells. Lipophagy is an autophagic process to select degrading lipid droplets. In yeast cells in stationary phase, vacuoles fuse and remodel their membranes to create a single spherical vacuole with two distinct membrane microdomains, which are required for yeast lipophagy. In this study, we discovered that Fat1p was capable of rapidly responding to changes in nutritional status and preserving cell survival by regulating membrane lipid saturation to maintain proper vacuolar morphology and the level of lipophagy in the yeast S. cerevisiae. Our findings shed light on how cells maintain vacuolar structure and promote the differentiation of vacuole surface microdomains for stationary-phase lipophagy.

Heterogeneity of Starved Yeast Cells in IF1 Levels Suggests the Role of This Protein in vivo.

In mitochondria, a small protein IF1 suppresses the hydrolytic activity of ATP synthase and presumably prevents excessive ATP hydrolysis under conditions of energy deprivation. In yeast Saccharomyces cerevisiae, IF1 homologs are encoded by two paralogous genes: INH1 and STF1. INH1 expression is known to aggravate the deleterious effects of mitochondrial DNA (mtDNA) depletion. Surprisingly, no beneficial effects of INH1 and STF1 were documented for yeast so far, and the functions of INH1 and STF1 in wild type cells are unclear. Here, we put forward a hypothesis that INH1 and STF1 bring advantage during the fast start of proliferation after reentry into exponential growth from post-diauxic or stationary phases. We found that yeast cells increase the concentration of both proteins in the post-diauxic phase. Post-diauxic phase yeast cells formed two subpopulations distinct in Inh1p and Stf1p concentrations. Upon exit from the post-diauxic phase cells with high level of Inh1-GFP started growing earlier than cells devoid of Inh1-GFP. However, double deletion of INH1 and STF1 did not increase the lag period necessary for stationary phase yeast cells to start growing after reinoculation into the fresh medium. These results point to a redundancy of the mechanisms preventing uncontrolled ATP hydrolysis during energy deprivation.

Pan2-Pan3 complex, together with Ccr4-Not complex, has a role in the cell growth on non-fermentable carbon sources

There are two major deadenylase complexes, Ccr4-Not and Pan2-Pan3, which shorten the 3′ poly(A) tail of mRNA and are conserved from yeast to human. We have previously shown that the Ccr4-mediated deadenylation plays the important role in gene expression regulation in the yeast stationary phase cell. In order to further understand the role of deadenylases in different growth condition, in this study we investigated the effect of deletion of both deadenylases on the cell in non-fermentable carbon containing media. We found that both ccr4Δ and ccr4Δ pan2Δ mutants showed similar growth defect in YPD media: when switched to media containing non-fermentable source (Glycerol-Lactate) only the ccr4Δ grew while the ccr4Δ pan2Δ did not. Ccr4, Pan2, and Pan3 were phosphorylated in GlyLac medium, suggesting that the activities of Ccr4, Pan2, and Pan3 may be regulated by phosphorylation in response to change of carbon sources. To get insights how Ccr4 and Pan2 function in the cell growth in media containing non-fermentable source only, we isolated multicopy suppressors for the growth defect on YPGlyLac media of the ccr4Δ pan2Δ mutant and identified two genes, STM1 and REX2, which encode a ribosome-associated protein and a 3′-5′ RNA exonuclease, respectively. Our results suggest that the Pan2-Pan3 complex, together with the Ccr4-Not complex, has important roles in the growth on non-fermentable carbon sources.

Cryptococcus neoformans resists to drastic conditions by switching to viable but non-culturable cell phenotype.

Metabolically quiescent pathogens can persist in a viable non-replicating state for months or even years. For certain infectious diseases, such as tuberculosis, cryptococcosis, histoplasmosis, latent infection is a corollary of this dormant state, which has the risk for reactivation and clinical disease. During murine cryptococcosis and macrophage uptake, stress and host immunity induce Cryptococcus neoformans heterogeneity with the generation of a sub-population of yeasts that manifests a phenotype compatible with dormancy (low stress response, latency of growth). In this subpopulation, mitochondrial transcriptional activity is regulated and this phenotype has been considered as a hallmark of quiescence in stem cells. Based on these findings, we worked to reproduce this phenotype in vitro and then standardize the experimental conditions to consistently generate this dormancy in C. neoformans. We found that incubation of stationary phase yeasts (STAT) in nutriment limited conditions and hypoxia for 8 days (8D-HYPOx) was able to produced cells that mimic the phenotype obtained in vivo. In these conditions, mortality and/or apoptosis occurred in less than 5% of the yeasts compared to 30–40% of apoptotic or dead yeasts upon incubation in normoxia (8D-NORMOx). Yeasts in 8D-HYPOx harbored a lower stress response, delayed growth and less that 1% of culturability on agar plates, suggesting that these yeasts are viable but non culturable cells (VBNC). These VBNC were able to reactivate in the presence of pantothenic acid, a vitamin that is known to be involved in quorum sensing and a precursor of acetyl-CoA. Global metabolism of 8D-HYPOx cells showed some specific requirements and was globally shut down compared to 8D-NORMOx and STAT conditions. Mitochondrial analyses showed that the mitochondrial mass increased with mitochondria mostly depolarized in 8D-HYPOx compared to 8D-NORMox, with increased expression of mitochondrial genes. Proteomic and transcriptomic analyses of 8D-HYPOx revealed that the number of secreted proteins and transcripts detected also decreased compared to 8D-NORMOx and STAT, and the proteome, secretome and transcriptome harbored specific profiles that are engaged as soon as four days of incubation. Importantly, acetyl-CoA and the fatty acid pathway involving mitochondria are required for the generation and viability maintenance of VBNC. Altogether, these data show that we were able to generate for the first time VBNC phenotype in C. neoformans. This VBNC state is associated with a specific metabolism that should be further studied to understand dormancy/quiescence in this yeast.

Investigating the long-term stability of protein immunogen(s) for whole recombinant yeast-based vaccines.

Even today vaccine(s) remains a mainstay in combating infectious diseases. Many yeast-based vaccines are currently in different phases of clinical trials. Despite the encouraging results of whole recombinant yeast and yeast display, the systematic study assessing the long-term stability of protein antigen(s) in yeast cells is still missing. Therefore, in the present study, I investigate the stability of heterologous protein antigen in the cellular environment of Saccharomyces cerevisiae through Escherichia coli surface protein (major curlin or CsgA). Present biochemical data showed that the stationary-phase yeast cells were able to keep the antigen stable for almost 1 year when stored at 2°C-8°C and 23°C-25°C. Further, iTRAQ-based quantitative proteomics of yeast whole cell lysate showed that the level of heterologous fusion protein was low in cells stored at 23°C-25°C compared to those at 2°C-8°C. In the end, I also proposed a workable strategy to test the integrity or completeness of heterologous protein in the yeast cell. I believe that the observations made in the present study will be really encouraging for those interested in the development of a whole recombinant yeast-based vaccine(s).

Low doses of DNA damaging agents extend Saccharomyces cerevisiae chronological lifespan by promoting entry into quiescence

A variety of mild stresses have been shown to extend lifespan in diverse species through hormesis, which is a beneficial response to a stress or toxin that would cause a negative response at a higher exposure. Whether particular stresses induce hormesis can vary with genotype for a given species, and the underlying mechanisms of lifespan extension are only partly understood in most cases. We show that low doses of the DNA damaging or replication stress agents hydroxyurea, methyl methanesulfonate, 4-nitroquinoline 1-oxide, or Zeocin (a phleomycin derivative) lengthened chronological lifespan in Saccharomyces cerevisiae if cells were exposed during growth, but not if they were exposed during stationary phase. Treatment with these agents did not change mitochondrial activity, increase resistance to acetic acid, ethanol, or heat stress, and three of four treatments did not increase resistance to hydrogen peroxide. Stationary phase yeast populations consist of both quiescent and nonquiescent cells, and all four treatments increased the proportion of quiescent cells. Several mutant strains with deletions in genes that influence quiescence prevented Zeocin treatment from extending lifespan and from increasing the proportion of quiescent stationary phase cells. These data indicate that mild DNA damage stress can extend lifespan by promoting quiescence in the absence of mitohormesis or improved general stress responses that have been frequently associated with improved longevity in other cases of hormesis. Further study of the underlying mechanism may yield new insights into quiescence regulation that will be relevant to healthy aging.

Consequences of Cytochrome c Oxidase Assembly Defects for the Yeast Stationary Phase

Functional assembly of cytochrome c oxidase (COX) is essential for an intact mitochondrial respiratory chain, although the consequences of a loss of assembled COX at yeast stationary phase remain largely unexplored. Stationary phase cultures are a good model for terminally differentiated cells in humans, such as neurons. Our goal is to define the physiological changes in mitochondrial respiration and oxidative stress between exponential and stationary phases in a respiratory competent strain and a set of COX assembly mutants with defects in a variety of aspects related to enzyme assembly. These results further our understanding of mitochondrial changes in terminally differentiated cells and aging cells. In this study, we show that a wild‐type respiratory competent yeast strain at stationary phase is characterized by a decreased oxidative capacity, as seen by a reduction in the amount of assembled COX, which is supported by a decrease in protein levels of several COX assembly factors. Wild‐type cells also have a disrupted mitochondrial reticulum during stationary phase. Loss of assembled COX results in the decreased abundance of many mitochondrial proteins at stationary phase, which we show is likely due to decreased membrane potential and changes in mitophagy. In addition to an altered mitochondrial proteome, COX assembly mutants display unexpected changes in markers of cellular oxidative stress at stationary phase. Specifically, differences in distribution of Sod1 between exponential and stationary phases give insight on location of reactive oxygen species (ROS) in the cell. Our results suggest that mitochondria may not be a major source of ROS at stationary phase in cells lacking an intact respiratory chain. Overall, we have identified fundamental differences between exponential and stationary phase mitochondria in respiratory competent yeast and have defined two distinct phenotypes, decreased levels of mitochondrial proteins and less mitochondrial oxidative damage, in cells with a defective respiratory chain. These findings suggest that COX deficiencies are associated with broader cellular phenotypes in terminally differentiated cells in humans.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada (NSERC)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Consequences of cytochrome c oxidase assembly defects for the yeast stationary phase

The assembly of cytochrome c oxidase (COX) is essential for a functional mitochondrial respiratory chain, although the consequences of a loss of assembled COX at yeast stationary phase, an excellent model for terminally differentiated cells in humans, remain largely unexamined. In this study, we show that a wild-type respiratory competent yeast strain at stationary phase is characterized by a decreased oxidative capacity, as seen by a reduction in the amount of assembled COX and by a decrease in protein levels of several COX assembly factors. In contrast, loss of assembled COX results in the decreased abundance of many mitochondrial proteins at stationary phase, which is likely due to decreased membrane potential and changes in mitophagy. In addition to an altered mitochondrial proteome, COX assembly mutants display unexpected changes in markers of cellular oxidative stress at stationary phase. Our results suggest that mitochondria may not be a major source of reactive oxygen species at stationary phase in cells lacking an intact respiratory chain.

Enhanced cell-surface display of a heterologous protein using SED1 anchoring system in SED1-disrupted Saccharomyces cerevisiae strain

Yeast displaying enzymes on the cell surface are used for developing whole-cell biocatalysts. High enzyme activity on the cell surface is required in certain applications such as direct ethanol production from lignocellulosic materials. However, the cell surface enzyme activity is limited by several factors, one of which is the protein amount of the yeast cell wall. In this study, we attempted to improve the incorporation capacity of a displayed heterologous enzyme by disrupting a native cell-wall protein. β-Glucosidase (BGL1) from Aspergillus aculeatus was fused with Saccharomyces cerevisiae Sed1 and displayed on the cell surface of S.cerevisiae BY4741 strain and its SED1 disruptant. Sed1 is one of the most abundant stationary phase yeast cell wall protein. A time course analysis revealed that BGL1 activity of the control strain reached saturation after 48h of cultivation. In contrast, the BGL1 activity of the SED1 disruptant increased until 72h of cultivation and was 22% higher than that of the control strain. We also performed relative quantification of cell wall proteins of these strains by nanoscale ultra pressure liquid chromatography electrospray ionization quadrupole time-of-flight tandem mass spectrometry (nano-UPLC-MSE). The amount of the cell wall-associated BGL1 per unit dry cell-weight of the SED1 disruptant was 19% higher than that of the control strain. These results suggested that the incorporation capacity of the cell wall for BGL1 was increased by disruption of SED1. Disruption of SED1 would be a promising approach for improving display efficiency of heterologous protein fused with Sed1.

Alpha-ketoglutarate enhances freeze-thaw tolerance and prevents carbohydrate-induced cell death of the yeast Saccharomyces cerevisiae.

Stress resistance and fermentative capability are important quality characteristics of baker's yeast. In the present study, we examined protective effects of exogenous alpha-ketoglutarate (AKG), an intermediate of the tricarboxylic acid cycle and amino acid metabolism, against freeze-thaw and carbohydrate-induced stresses in the yeast Saccharomyces cerevisiae. Growth on AKG-supplemented medium prevented a loss of viability and improved fermentative capacity of yeast cells after freeze-thaw treatment. The cells grown in the presence of AKG had higher levels of amino acids (e.g., proline), higher metabolic activity and total antioxidant capacity, and higher activities of catalase, NADP-dependent glutamate dehydrogenase and glutamine synthase compared to control ones. Both synthesis of amino acids and enhancement of antioxidant system capacity could be involved in AKG-improved freeze-thaw tolerance in S. cerevisiae. Cell viability dramatically decreased under incubation of stationary-phase yeast cells in 2% glucose or fructose solutions (in the absence of the other nutrients) as compared with incubation in distilled water or in 10mM AKG solution. The decrease in cell viability was accompanied by acidification of the medium, and decrease in cellular respiration, aconitase activity, and levels of total protein and free amino acids. The supplementation with 10mM AKG effectively prevented carbohydrate-induced yeast death. Protective mechanisms of AKG could be associated with the intensification of respiration and prevention of decreasing protein level as well as with direct antioxidant AKG action.

The control of translational accuracy is a determinant of healthy ageing in yeast.

Life requires the maintenance of molecular function in the face of stochastic processes that tend to adversely affect macromolecular integrity. This is particularly relevant during ageing, as many cellular functions decline with age, including growth, mitochondrial function and energy metabolism. Protein synthesis must deliver functional proteins at all times, implying that the effects of protein synthesis errors like amino acid misincorporation and stop-codon read-through must be minimized during ageing. Here we show that loss of translational accuracy accelerates the loss of viability in stationary phase yeast. Since reduced translational accuracy also reduces the folding competence of at least some proteins, we hypothesize that negative interactions between translational errors and age-related protein damage together overwhelm the cellular chaperone network. We further show that multiple cellular signalling networks control basal error rates in yeast cells, including a ROS signal controlled by mitochondrial activity, and the Ras pathway. Together, our findings indicate that signalling pathways regulating growth, protein homeostasis and energy metabolism may jointly safeguard accurate protein synthesis during healthy ageing.

Removal of Ochratoxin A in Saccharomyces cerevisiae Liquid Cultures

The capacity for removal of ochratoxin A (OTA) during alcoholic fermentation was evaluated in batch systems with one commercial strain and one wild strain of Saccharomyces cerevisiae. Batch alcoholic fermentations were carried out in yeast extract-malt extract broth (YM) medium, with 18.0% glucose and OTA added to final concentrations of 3.48 and 4.95 ng/mL respectively. The removal capacity of each yeast strain was examined after completion of fermentation in batch culture and after extended contact with yeast biomass. The removal capacity of the yeast strains was also examined in stationary phase cultures. Stationary phase yeasts were studied with biomass harvested from the stationary phase of anaerobic fermentation, by incubation in phosphate buffer, with the addition of 5.00 ng/ mL of OTA. Removal studies with stationary phase cells were performed with viable and non-viable cells inactivated with Na-azide. The study showed that in growing phase cultures, OTA removal was significant only after extended contact with yeast biomass; up to 29.7% and 25.4% for wild yeast ZIM 1927 and commercial yeast Lalvin EC-1118 respectively, but not during alcoholic fermentation. In stationary phase cultures, viable and non-viable cells were not significantly different in OTA removal from the medium. This demonstrated that OTA was not metabolised, but possibly adsorbed by the yeast cells. The presence of OTA in synthetic media influenced yeast metabolism, causing the production of higher volatile acidity by 0.08 and 0.13 g/L for Lalvin EC-1118 and ZIM 1927 respectively, and lower concentrations of reducing sugar, by 0.32 g/L, but only for ZIM 1927.

The glyoxylate shunt is essential for desiccation tolerance in C. elegans and budding yeast.

Many organisms, including species from all kingdoms of life, can survive desiccation by entering a state with no detectable metabolism. To survive, C. elegans dauer larvae and stationary phase S. cerevisiae require elevated amounts of the disaccharide trehalose. We found that dauer larvae and stationary phase yeast switched into a gluconeogenic mode in which metabolism was reoriented toward production of sugars from non-carbohydrate sources. This mode depended on full activity of the glyoxylate shunt (GS), which enables synthesis of trehalose from acetate. The GS was especially critical during preparation of worms for harsh desiccation (preconditioning) and during the entry of yeast into stationary phase. Loss of the GS dramatically decreased desiccation tolerance in both organisms. Our results reveal a novel physiological role for the GS and elucidate a conserved metabolic rewiring that confers desiccation tolerance on organisms as diverse as worm and yeast.

Differentiated cytoplasmic granule formation in quiescent and non-quiescent cells upon chronological aging.

Stationary phase cultures represent a complicated cell population comprising at least two different cell types, quiescent (Q) and non-quiescent (NQ) cells. Q and NQ cells have different lifespans and cell physiologies. However, less is known about the organization of cytosolic protein structures in these two cell types. In this study, we examined Q and NQ cells for the formation of several stationary phase-prevalent granule structures including actin bodies, proteasome storage granules, stress granules, P-bodies, the compartment for unconventional protein secretion (CUPS), and Hsp42-associated stationary phase granules (Hsp42-SPGs). Most of these structures preferentially form in NQ cells, except for Hsp42-SPGs, which are enriched in Q cells. When nutrients are provided, NQ cells enter mitosis less efficiently than Q cells, likely due to the time requirement for reorganizing some granule structures. We observed that heat shock-induced misfolded proteins often colocalize to Hsp42-SPGs, and Q cells clear these protein aggregates more efficiently, suggesting that Hsp42-SPGs may play an important role in the stress resistance of Q cells. Finally, we show that the cell fate of NQ cells is largely irreversible even if they are allowed to reenter mitosis. Our results reveal that the formation of different granule structures may represent the early stage of cell type differentiation in yeast stationary phase cultures.

A haploproficient interaction of the transaldolase paralogue NQM1 with the transcription factor VHR1 affects stationary phase survival and oxidative stress resistance.

BackgroundStudying the survival of yeast in stationary phase, known as chronological lifespan, led to the identification of molecular ageing factors conserved from yeast to higher organisms. To identify functional interactions among yeast chronological ageing genes, we conducted a haploproficiency screen on the basis of previously identified long-living mutants. For this, we created a library of heterozygous Saccharomyces cerevisiae double deletion strains and aged them in a competitive manner.ResultsStationary phase survival was prolonged in a double heterozygous mutant of the metabolic enzyme non-quiescent mutant 1 (NQM1), a paralogue to the pentose phosphate pathway enzyme transaldolase (TAL1), and the transcription factor vitamin H response transcription factor 1 (VHR1). We find that cells deleted for the two genes possess increased clonogenicity at late stages of stationary phase survival, but find no indication that the mutations delay initial mortality upon reaching stationary phase, canonically defined as an extension of chronological lifespan. We show that both genes influence the concentration of metabolites of glycolysis and the pentose phosphate pathway, central metabolic players in the ageing process, and affect osmolality of growth media in stationary phase cultures. Moreover, NQM1 is glucose repressed and induced in a VHR1 dependent manner upon caloric restriction, on non-fermentable carbon sources, as well as under osmotic and oxidative stress. Finally, deletion of NQM1 is shown to confer resistance to oxidizing substances.ConclusionsThe transaldolase paralogue NQM1 and the transcription factor VHR1 interact haploproficiently and affect yeast stationary phase survival. The glucose repressed NQM1 gene is induced under various stress conditions, affects stress resistance and this process is dependent on VHR1. While NQM1 appears not to function in the pentose phosphate pathway, the interplay of NQM1 with VHR1 influences the yeast metabolic homeostasis and stress tolerance during stationary phase, processes associated with yeast ageing.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-015-0171-6) contains supplementary material, which is available to authorized users.

Cell cycle-independent phospho-regulation of Fkh2 during hyphal growth regulates Candida albicans pathogenesis.

The opportunistic human fungal pathogen, Candida albicans, undergoes morphological and transcriptional adaptation in the switch from commensalism to pathogenicity. Although previous gene-knockout studies have identified many factors involved in this transformation, it remains unclear how these factors are regulated to coordinate the switch. Investigating morphogenetic control by post-translational phosphorylation has generated important regulatory insights into this process, especially focusing on coordinated control by the cyclin-dependent kinase Cdc28. Here we have identified the Fkh2 transcription factor as a regulatory target of both Cdc28 and the cell wall biosynthesis kinase Cbk1, in a role distinct from its conserved function in cell cycle progression. In stationary phase yeast cells 2D gel electrophoresis shows that there is a diverse pool of Fkh2 phospho-isoforms. For a short window on hyphal induction, far before START in the cell cycle, the phosphorylation profile is transformed before reverting to the yeast profile. This transformation does not occur when stationary phase cells are reinoculated into fresh medium supporting yeast growth. Mass spectrometry and mutational analyses identified residues phosphorylated by Cdc28 and Cbk1. Substitution of these residues with non-phosphorylatable alanine altered the yeast phosphorylation profile and abrogated the characteristic transformation to the hyphal profile. Transcript profiling of the phosphorylation site mutant revealed that the hyphal phosphorylation profile is required for the expression of genes involved in pathogenesis, host interaction and biofilm formation. We confirmed that these changes in gene expression resulted in corresponding defects in pathogenic processes. Furthermore, we identified that Fkh2 interacts with the chromatin modifier Pob3 in a phosphorylation-dependent manner, thereby providing a possible mechanism by which the phosphorylation of Fkh2 regulates its specificity. Thus, we have discovered a novel cell cycle-independent phospho-regulatory event that subverts a key component of the cell cycle machinery to a role in the switch from commensalism to pathogenicity.

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 long-term 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.

Deteriorated stress response in stationary-phase yeast: Sir2 and Yap1 are essential for Hsf1 activation by heat shock and oxidative stress, respectively.

Stationary-phase cultures have been used as an important model of aging, a complex process involving multiple pathways and signaling networks. However, the molecular processes underlying stress response of non-dividing cells are poorly understood, although deteriorated stress response is one of the hallmarks of aging. The budding yeast Saccharomyces cerevisiae is a valuable model organism to study the genetics of aging, because yeast ages within days and are amenable to genetic manipulations. As a unicellular organism, yeast has evolved robust systems to respond to environmental challenges. This response is orchestrated largely by the conserved transcription factor Hsf1, which in S. cerevisiae regulates expression of multiple genes in response to diverse stresses. Here we demonstrate that Hsf1 response to heat shock and oxidative stress deteriorates during yeast transition from exponential growth to stationary-phase, whereas Hsf1 activation by glucose starvation is maintained. Overexpressing Hsf1 does not significantly improve heat shock response, indicating that Hsf1 dwindling is not the major cause for Hsf1 attenuated response in stationary-phase yeast. Rather, factors that participate in Hsf1 activation appear to be compromised. We uncover two factors, Yap1 and Sir2, which discretely function in Hsf1 activation by oxidative stress and heat shock. In Δyap1 mutant, Hsf1 does not respond to oxidative stress, while in Δsir2 mutant, Hsf1 does not respond to heat shock. Moreover, excess Sir2 mimics the heat shock response. This role of the NAD+-dependent Sir2 is supported by our finding that supplementing NAD+ precursors improves Hsf1 heat shock response in stationary-phase yeast, especially when combined with expression of excess Sir2. Finally, the combination of excess Hsf1, excess Sir2 and NAD+ precursors rejuvenates the heat shock response.

SOD1 oxidation and formation of soluble aggregates in yeast: Relevance to sporadic ALS development

Misfolding and aggregation of copper–zinc superoxide dismutase (Sod1) are observed in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Mutations in Sod1 lead to familial ALS (FALS), which is a late-onset disease. Since oxidative damage to proteins increases with age, it had been proposed that oxidation of Sod1 mutants may trigger their misfolding and aggregation in FALS. However, over 90% of ALS cases are sporadic (SALS) with no obvious genetic component. We hypothesized that oxidation could also trigger the misfolding and aggregation of wild-type Sod1 and sought to confirm this in a cellular environment. Using quiescent, stationary-phase yeast cells as a model for non-dividing motor neurons, we probed for post-translational modification (PTM) and aggregation of wild-type Sod1 extracted from these cells. By size-exclusion chromatography (SEC), we isolated two populations of Sod1 from yeast: a low-molecular weight (LMW) fraction that is catalytically active and a catalytically inactive, high-molecular weight (HMW) fraction. High-resolution mass spectrometric analysis revealed that LMW Sod1 displays no PTMs but HMW Sod1 is oxidized at Cys146 and His71, two critical residues for the stability and folding of the enzyme. HMW Sod1 is also oxidized at His120, a copper ligand, which will promote loss of this catalytic metal cofactor essential for SOD activity. Monitoring the fluorescence of a Sod1-green-fluorescent-protein fusion (Sod1-GFP) extracted from yeast chromosomally expressing this fusion, we find that HMW Sod1-GFP levels increase up to 40-fold in old cells. Thus, we speculate that increased misfolding and inclusion into soluble aggregates is a consequence of elevated oxidative modifications of wild-type Sod1 as cells age. Our observations argue that oxidative damage to wild-type Sod1 initiates the protein misfolding mechanisms that give rise to SALS.