Autophagy proteins play cytoprotective and cytocidal roles in leucine starvation-induced cell death in Saccharomyces cerevisiae

Autophagy is essential for prolonging yeast survival during nutrient deprivation; however, this report shows that some autophagy proteins may also be accelerating population death in those conditions. While leucine starvation caused YCA1-mediated apoptosis characterized by increased annexin V staining, nitrogen deprivation triggered necrotic death characterized by increased propidium iodide uptake. Although a Δatg8 strain died faster than its parental strain during nitrogen starvation, this mutant died slower than its parent during leucine starvation. Conversely, a Δatg11 strain died slower than its parent during nitrogen starvation, but faster during leucine starvation. Curiously, although GFP-Atg8 complemented the Δatg8 mutation, this protein made ATG8 cells more sensitive to nitrogen starvation, and less sensitive to leucine starvation. These results were difficult to explain if autophagy only extended life but could be an indication that a second form of autophagy could concurrently facilitate either apoptotic or necrotic cell death.     

Autophagy proteins play cytoprotective and cytocidal roles in leucine starvation-induced cell death in Saccharomyces cerevisiae

Autophagy is essential for prolonging yeast survival during nutrient deprivation; however, this report shows that some autophagy proteins may also be accelerating population death in those conditions. While leucine starvation caused YCA1-mediated apoptosis characterized by increased annexin V staining, nitrogen deprivation triggered necrotic death characterized by increased propidium iodide uptake. Although a Δatg8 strain died faster than its parental strain during nitrogen starvation, this mutant died slower than its parent during leucine starvation. Conversely, a Δatg11 strain died slower than its parent during nitrogen starvation, but faster during leucine starvation. Curiously, although GFP-Atg8 complemented the Δatg8 mutation, this protein made ATG8 cells more sensitive to nitrogen starvation, and less sensitive to leucine starvation. These results were difficult to explain if autophagy only extended life but could be an indication that a second form of autophagy could concurrently facilitate either apoptotic or necrotic cell death.     

Vanadate-resistant mutants of Candida albicans show alterations in phosphate uptake

Phosphate uptake studies in different strains of the dimorphic pathogenic yeast Candida albicans were undertaken to show that this yeast actively transported phosphate with an apparent Km in the range of 90-170 microM. The uptake was pH dependent and derepressible under phosphate starvation. Vanadate-resistant (van) mutants of C. albicans showed a 20-70% reduction in the rate of phosphate uptake in high phosphate medium and was associated with an increased Km and reduced Vmax. The magnitude of derepression under phosphate starvation was different between van mutants. These results demonstrate that van mutants may have developed resistance by modifying the rate of entry of vanadate.     

Nucleotide degradation and ribose salvage in yeast

Nucleotide degradation is a universal metabolic capability. Here we combine metabolomics, genetics and biochemistry to characterize the yeast pathway. Nutrient starvation, via PKA, AMPK/SNF1, and TOR, triggers autophagic breakdown of ribosomes into nucleotides. A protein not previously associated with nucleotide degradation, Phm8, converts nucleotide monophosphates into nucleosides. Downstream steps, which involve the purine nucleoside phosphorylase, Pnp1, and pyrimidine nucleoside hydrolase, Urh1, funnel ribose into the nonoxidative pentose phosphate pathway. During carbon starvation, the ribose‐derived carbon accumulates as sedoheptulose‐7‐phosphate, whose consumption by transaldolase is impaired due to depletion of transaldolase's other substrate, glyceraldehyde‐3‐phosphate. Oxidative stress increases glyceraldehyde‐3‐phosphate, resulting in rapid consumption of sedoheptulose‐7‐phosphate to make NADPH for antioxidant defense. Ablation of Phm8 or double deletion of Pnp1 and Urh1 prevent effective nucleotide salvage, resulting in metabolite depletion and impaired survival of starving yeast. Thus, ribose salvage provides means of surviving nutrient starvation and oxidative stress.     

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.     

Intracellular protein breakdown in non-growing cells of Escherichia coli

1. When Escherichia coli leu(-) was incubated at 35 degrees in a medium based on minimal medium, but with the omission of phosphate ions, or glucose, or NH(4) (+) ions and leucine, intracellular protein was degraded at a rate of about 5%/hr. in each case. If Mg(2+) ions were omitted, however, the rate of degradation was 2.9%/hr. 2. Under certain conditions of incubation, protein degradation was inhibited. The inhibitor was neither NH(4) (+) ions nor amino acids, and its properties were not those of a protein, but it might be an unstable species of RNA. 3. Although a large part of the cell protein was degraded at about 5%/hr. during starvation of NH(4) (+) ions and leucine, some proteins were lost at more rapid rates, whereas others were lost at lower rates or not at all. 4. In particular, beta-galactosidase activity was lost at about 8%/hr. during starvation of NH(4) (+) ions and leucine, whereas d-serine-deaminase and alkaline-phosphatase activities were stable. During starvation of Mg(2+) ions, all three enzyme activities were stable.     

Attenuation of phosphate starvation responses by phosphite in Arabidopsis.

When inorganic phosphate is limiting, Arabidopsis has the facultative ability to metabolize exogenous nucleic acid substrates, which we utilized previously to identify insensitive phosphate starvation response mutants in a conditional genetic screen. In this study, we examined the effect of the phosphate analog, phosphite (Phi), on molecular and morphological responses to phosphate starvation. Phi significantly inhibited plant growth on phosphate-sufficient (2 mM) and nucleic acid-containing (2 mM phosphorus) media at concentrations higher than 2.5 mM. However, with respect to suppressing typical responses to phosphate limitation, Phi effects were very similar to those of phosphate. Phosphate starvation responses, which we examined and found to be almost identically affected by both anions, included changes in: (a) the root-to-shoot ratio; (b) root hair formation; (c) anthocyanin accumulation; (d) the activities of phosphate starvation-inducible nucleolytic enzymes, including ribonuclease, phosphodiesterase, and acid phosphatase; and (e) steady-state mRNA levels of phosphate starvation-inducible genes. It is important that induction of primary auxin response genes by indole-3-acetic acid in the presence of growth-inhibitory Phi concentrations suggests that Phi selectively inhibits phosphate starvation responses. Thus, the use of Phi may allow further dissection of phosphate signaling by genetic selection for constitutive phosphate starvation response mutants on media containing organophosphates as the only source of phosphorus.     

A pheromone mutant of Schizosaccharomyces pombe displays nucleolar fragmentation

Stresses and nutritional starvation are two main external signals for the induction of sex pheromones in the fission yeast Schizosaccharomyces pombe. In an attempt to identify the components involved in transduction of starvation signals, we screened 135 temperature-sensitive (ts) mutants and isolated 6 mutants that induced the pheromone even in the presence of a nitrogen source. These mutants exhibited two distinct induction phenotypes: pheromone induction at restrictive but not at permissive temperatures; and pheromone induction at both permissive and restrictive temperatures. The times required for the maximum pheromone induction at the restrictive temperature differed slightly in each mutant. In addition to the pheromone induction phenotype, the ts243 and ts304 mutants exhibited cell-division-cycle defects. The ts304 mutant cells showed an abnormal cytoplasmic DAPI staining pattern. The nucleolus of this mutant seemed to be fragmented, a phenomenon which is typically observed in aged yeast cells. The result of our genetic analysis indicated that the pheromone induction mutants belonged to 6 separate complementation groups. We designated these mutants pws1 to pws6.     

RpoS is necessary for both the positive and negative regulation of starvation survival genes during phosphate, carbon, and nitrogen starvation in Salmonella typhimurium.

The starvation stress response of Salmonella typhimurium encompasses the genetic and physiologic changes that occur when this bacterium is starved for an essential nutrient such as phosphate (P), carbon (C), or nitrogen (N). The responses to the limitation of each of these nutrients involve both unique and overlapping sets of proteins important for starvation survival and virulence. The role of the alternative sigma factor RpoS in the regulation of the starvation survival loci, stiA, stiB, and stiC, has been characterized. RpoS (sigma S) was found to be required for the P, C, and N starvation induction of stiA and stiC. In contrast, RpoS was found to be required for the negative regulation of stiB during P and C starvation-induced stationary phase but not during logarithmic phase. This role was independent of the relA gene (previously found to be needed for stiB induction). The role of RpoS alone and in combination with one or more sti mutations in the starvation survival of the organism was also investigated. The results clearly demonstrate that RpoS is an integral component of the complex interconnected regulatory systems involved in S. typhimurium's response to nutrient deprivation. However, differential responses of various sti genes indicate that additional signals and regulatory proteins are also involved.     

Differential regulation by cyclic AMP of starvation protein synthesis in Escherichia coli

Of the 30 carbon starvation proteins whose induction has been previously shown to be important for starvation survival of Escherichia coli, two-thirds were not induced in cya or crp deletion mutants of E. coli at the onset of carbon starvation. The rest were induced, although not necessarily with the same temporal pattern as exhibited in the wild type. The starvation proteins that were homologous to previously identified heat shock proteins belonged to the latter class and were hyperinduced in delta cya or delta crp mutants during starvation. Most of the cyclic AMP-dependent proteins were synthesized in the delta cya mutant if exogenous cyclic AMP was added at the onset of starvation. Furthermore, beta-galactosidase induction of several carbon starvation response gene fusions occurred only in a cya+ genetic background. Thus, two-thirds of the carbon starvation proteins of E. coli require cyclic AMP and its receptor protein for induction; the rest do not. The former class evidently has no role in starvation survival, since delta cya or delta crp mutants of either E. coli or Salmonella typhimurium survived starvation as well as their wild-type parents did. The latter class, therefore, is likely to have a direct role in starvation survival. This possibility is strengthened by the finding that nearly all of the cya- and crp-independent proteins were also induced during nitrogen starvation and, as shown previously, during phosphate starvation. Proteins whose synthesis is independent of cya- and crp control are referred to as Pex (postexponential).     

Genetic and genomic evidence that sucrose is a global regulator of plant responses to phosphate starvation in Arabidopsis

Plants respond to phosphate (Pi) starvation by exhibiting a suite of developmental, biochemical, and physiological changes to cope with this nutritional stress. To understand the molecular mechanism underlying these responses, we isolated an Arabidopsis (Arabidopsis thaliana) mutant, hypersensitive to phosphate starvation1 (hps1), which has enhanced sensitivity in almost all aspects of plant responses to Pi starvation. Molecular and genetic analyses indicated that the mutant phenotype is caused by overexpression of the SUCROSE TRANSPORTER2 (SUC2) gene. As a consequence, hps1 has a high level of sucrose (Suc) in both its shoot and root tissues. Overexpression of SUC2 or its closely related family members SUC1 and SUC5 in wild-type plants recapitulates the phenotype of hps1. In contrast, the disruption of SUC2 functions greatly inhibits plant responses to Pi starvation. Microarray analysis further indicated that 73% of the genes that are induced by Pi starvation in wild-type plants can be induced by elevated levels of Suc in hps1 mutants, even when they are grown under Pi-sufficient conditions. These genes include several important Pi signaling components and those that are directly involved in Pi transport, mobilization, and distribution between shoot and root. Interestingly, Suc and low-Pi signals appear to interact with each other both synergistically and antagonistically in regulating gene expression. Our genetic and genomic studies provide compelling evidence that Suc is a global regulator of plant responses to Pi starvation. This finding will help to further elucidate the signaling mechanism that controls plant responses to this particular nutritional stress.     

Chemotactic Responses of Marine Vibrio sp. Strain S14 (CCUG 15956) to Low-Molecular-Weight Substances under Starvation and Recovery Conditions

The chemotactic responses by starved cells of marine Vibrio sp. strain S14 differed from those elicited by cells that were not nutrient limited. The rate of chemotaxis at different concentrations of several attractants varied for starved and growing cells. Vibrio sp. strain S14 showed positive chemotaxis to leucine, valine, arginine, and glucose at the onset of energy and nutrient deprivation. A continued, though decreased, positive response was demonstrated fro leucine, arginine, and glucose at 10 h of starvation. Cells starved for 3 h displayed a stronger response to glucose than those starved for shorter or longer times. However, cells starved for 5 and 10 h responded more strongly to a lower concentration of glucose than did cells starved for 0 and 3 h. Starvation for 24 h elicited no measurable chemotaxis to leucine, arginine, or glucose. The motility decreased by over 95% in the cell population after 24 h of starvation, which resulted in a low sensitivity in the chemotaxis assay. A switch in the response to valine was observed by 3 h of starvation. The addition of nutrients of 22-h-starved cells elicited a temporary positive chemotactic response to leucine by 2 and 4 h of nutrient recovery, while cells at 1 and 6 h of recovery showed no response. At 2 h of recovery, the greatest response was recorded to 10⁻⁴ M leucine, whereas at 4 h it was to 10⁻² M leucine. Ten to fifty percent of the 22-h-starved cell population regained their motility after 4 h of nutrient-aided recovery. It is possible that two types of chemosensory systems exist in marine bacteria. Starved and growing cells responded to different concentrations of the attractant, and growing cells displayed a saturated chemotactic system with leucine as the attractant, unlike the response during starvation.     

Acetic acid inhibits nutrient uptake in Saccharomyces cerevisiae: auxotrophy confounds the use of yeast deletion libraries for strain improvement

Acetic acid inhibition of yeast fermentation has a negative impact in several industrial processes. As an initial step in the construction of a Saccharomyces cerevisiae strain with increased tolerance for acetic acid, mutations conferring resistance were identified by screening a library of deletion mutants in a multiply auxotrophic genetic background. Of the 23 identified mutations, 11 were then introduced into a prototrophic laboratory strain for further evaluation. Because none of the 11 mutations was found to increase resistance in the prototrophic strain, potential interference by the auxotrophic mutations themselves was investigated. Mutants carrying single auxotrophic mutations were constructed and found to be more sensitive to growth inhibition by acetic acid than an otherwise isogenic prototrophic strain. At a concentration of 80 mM acetic acid at pH 4.8, the initial uptake of uracil, leucine, lysine, histidine, tryptophan, phosphate, and glucose was lower in the prototrophic strain than in a non-acetic acid-treated control. These findings are consistent with two mechanisms by which nutrient uptake may be inhibited. Intracellular adenosine triphosphate (ATP) levels were severely decreased upon acetic acid treatment, which likely slowed ATP-dependent proton symport, the major form of transport in yeast for nutrients other than glucose. In addition, the expression of genes encoding some nutrient transporters was repressed by acetic acid, including HXT1 and HXT3 that encode glucose transporters that operate by facilitated diffusion. These results illustrate how commonly used genetic markers in yeast deletion libraries complicate the effort to isolate strains with increased acetic acid resistance.     

Starvation-Associated Genome Restructuring Can Lead to Reproductive Isolation in Yeast

Knowledge of the mechanisms that lead to reproductive isolation is essential for understanding population structure and speciation. While several models have been advanced to explain post-mating reproductive isolation, experimental data supporting most are indirect. Laboratory investigations of this phenomenon are typically carried out under benign conditions, which result in low rates of genetic change unlikely to initiate reproductive isolation. Previously, we described an experimental system using the yeast Saccharomyces cerevisiae where starvation served as a proxy to any stress that decreases reproduction and/or survivorship. We showed that novel lineages with restructured genomes quickly emerged in starved populations, and that these survivors were more fit than their ancestors when re-starved. Here we show that certain yeast lineages that survive starvation have become reproductively isolated from their ancestor. We further demonstrate that reproductive isolation arises from genomic rearrangements, whose frequency in starving yeast is several orders of magnitude greater than an unstarved control. By contrast, the frequency of point mutations is less than 2-fold greater. In a particular case, we observe that a starved lineage becomes reproductively isolated as a direct result of the stress-related accumulation of a single chromosome. We recapitulate this result by demonstrating that introducing an extra copy of one or several chromosomes into naïve, i.e. unstarved, yeast significantly diminishes their fertility. This type of reproductive barrier, whether arising spontaneously or via genetic manipulation, can be removed by making a lineage euploid for the altered chromosomes. Our model provides direct genetic evidence that reproductive isolation can arise frequently in stressed populations via genome restructuring without the precondition of geographic isolation.     

Biosynthesis of Branched-Chain Amino Acids in Yeast: Correlation of Biochemical Blocks and Genetic Lesions in Leucine Auxotrophs

The three enzymatic steps in the conversion of alpha-ketoisovalerate to alpha-ketoisocaproate were examined in wild-type and in leucine auxotrophic stocks of yeast. Procedures for the reliable assay of each of the enzymatic steps in crude extracts were devised. Crude extracts of the prototrophic haploid stock catalyzed all three enzymatic steps. Examination of a series of leucine auxotrophs permitted a correlation between the three enzymatic steps and the genetic lesions affecting 10 different loci. This examination revealed that a single locus (le-6) affected primarily alpha-isopropylmalate synthetase, the first step in the pathway. Lesions in six loci (le-1, le-4, le-5, le-7, le-8, and le-10) lead primarily to a deficiency in the activity of the second enzyme in the pathway, alpha-isopropylmalate isomerase. Stocks with lesions in three loci (le-2, le-3, and le-9) were primarily blocked in the third step of the pathway, catalyzed by beta-isopropylmalate dehydrogenase. The results with the mutants provide strong evidence that the pathway for leucine biosynthesis proposed by Strassman and his colleagues is the sole significant pathway in yeast.     

Phosphate homeostasis in the yeast Saccharomyces cerevisiae, the key role of the SPX domain-containing proteins

In the yeast Saccharomyces cerevisiae, a working model for nutrient homeostasis in eukaryotes, inorganic phosphate (Pi) homeostasis is regulated by the PHO pathway, a set of phosphate starvation induced genes, acting to optimize Pi uptake and utilization. Among these, a subset of proteins containing the SPX domain has been shown to be key regulators of Pi homeostasis. In this review, we summarize the recent progresses in elucidating the mechanisms controlling Pi homeostasis in yeast, focusing on the key roles of the SPX domain-containing proteins in these processes, as well as describing the future challenges and opportunities in this fast-moving field.     

High-Resolution Profiling of Stationary-Phase Survival Reveals Yeast Longevity Factors and Their Genetic Interactions

Lifespan is influenced by a large number of conserved proteins and gene-regulatory pathways. Here, we introduce a strategy for systematically finding such longevity factors in Saccharomyces cerevisiae and scoring the genetic interactions (epistasis) among these factors. Specifically, we developed an automated competition-based assay for chronological lifespan, defined as stationary-phase survival of yeast populations, and used it to phenotype over 5,600 single- or double-gene knockouts at unprecedented quantitative resolution. We found that 14% of the viable yeast mutant strains were affected in their stationary-phase survival; the extent of true-positive chronological lifespan factors was estimated by accounting for the effects of culture aeration and adaptive regrowth. We show that lifespan extension by dietary restriction depends on the Swr1 histone-exchange complex and that a functional link between autophagy and the lipid-homeostasis factor Arv1 has an impact on cellular lifespan. Importantly, we describe the first genetic interaction network based on aging phenotypes, which successfully recapitulated the core-autophagy machinery and confirmed a role of the human tumor suppressor PTEN homologue in yeast lifespan and phosphatidylinositol phosphate metabolism. Our quantitative analysis of longevity factors and their genetic interactions provides insights into the gene-network interactions of aging cells.