The Gap1 general amino acid permease acts as an amino acid sensor for activation of protein kinase A targets in the yeast Saccharomyces cerevisiae

Addition of a nitrogen source to yeast (Saccharomyces cerevisiae) cells starved for nitrogen on a glucose-containing medium triggers activation of protein kinase A (PKA) targets through a pathway that requires for sustained activation both a fermentable carbon source and a complete growth medium (fermentable growth medium induced or FGM pathway). Trehalase is activated, trehalose and glycogen content as well as heat resistance drop rapidly, STRE-controlled genes are repressed, and ribosomal protein genes are induced. We show that the rapid effect of amino acids on these targets specifically requires the general amino acid permease Gap1. In the gap1Delta strain, transport of high concentrations of l-citrulline occurs at a high rate but without activation of trehalase. Metabolism of the amino acids is not required. Point mutants in Gap1 with reduced or deficient transport also showed reduced or deficient signalling. However, two mutations, S391A and S397A, were identified with a differential effect on transport and signalling for l-glutamate and l-citrulline. Specific truncations of the C-terminus of Gap1 (e.g. last 14 or 26 amino acids) did not reduce transport activity but caused the same phenotype as in strains with constitutively high PKA activity also during growth with ammonium as sole nitrogen source. The overactive PKA phenotype was abolished by mutations in the Tpk1 or Tpk2 catalytic subunits. We conclude that Gap1 acts as an amino acid sensor for rapid activation of the FGM signalling pathway which controls the PKA targets, that transport through Gap1 is connected to signalling and that specific truncations of the C-terminus result in permanently activating Gap1 alleles.     

The Ras/cAMP-dependent protein kinase signaling pathway regulates an early step of the autophagy process in Saccharomyces cerevisiae

When faced with nutrient deprivation, Saccharomyces cerevisiae cells enter into a nondividing resting state, known as stationary phase. The Ras/PKA (cAMP-dependent protein kinase) signaling pathway plays an important role in regulating the entry into this resting state and the subsequent survival of stationary phase cells. The survival of these resting cells is also dependent upon autophagy, a membrane trafficking pathway that is induced upon nutrient deprivation. Autophagy is responsible for targeting bulk protein and other cytoplasmic constituents to the vacuolar compartment for their ultimate degradation. The data presented here demonstrate that the Ras/PKA signaling pathway inhibits an early step in autophagy because mutants with elevated levels of Ras/PKA activity fail to accumulate transport intermediates normally associated with this process. Quantitative assays indicate that these increased levels of Ras/PKA signaling activity result in an essentially complete block to autophagy. Interestingly, Ras/PKA activity also inhibited a related process, the cytoplasm to vacuole targeting (Cvt) pathway that is responsible for the delivery of a subset of vacuolar proteins in growing cells. These data therefore indicate that the Ras/PKA signaling pathway is not regulating a switch between the autophagy and Cvt modes of transport. Instead, it is more likely that this signaling pathway is controlling an activity that is required during the early stages of both of these membrane trafficking pathways. Finally, the data suggest that at least a portion of the Ras/PKA effects on stationary phase survival are the result of the regulation of autophagy activity by this signaling pathway.     

Role for YakA,cAMP, and protein kinase A in regulation of stress responses of Dictyostelium discoideum cells

The Dictyostelium protein kinase YakA is required for the growth-to-development transition. During growth YakA controls the cell cycle, regulating the intervals between cell divisions. When starved for nutrients Dictyostelium cells arrest growth and undergo changes in gene expression, decreasing vegetative mRNAs and inducing the expression of pkaC. YakA is an effector of these changes, being necessary for the decrease of vegetative mRNA expression and the increase of protein kinase A (PKA) activity that will ultimately regulate expression of adenylyl cyclase, cAMP synthesis, and the induction of development. We report a role for this kinase in the response to nitrosoative or oxidative stress of Dictyostelium cells. Hydrogen peroxide and sodium nitroprusside arrest the growth of cells and trigger cAMP synthesis and activation of PKA in a manner similar to the well-established response to nutrient starvation. We have found that yakA null cells are hypersensitive to nitrosoative/oxidative stress and that a second-site mutation in pkaC suppresses this sensitivity. The response to different stresses has been investigated and YakA, cAMP, and PKA have been identified as components of the pathway that regulate the growth arrest that follows treatment with compounds that generate reactive oxygen species. The effect of different types of stress was evaluated in Dictyostelium and the YakA/PKA pathway was also implicated in the response to heat stress.     

In Vivo Phosphorylation of Ser21 and Ser83 during Nutrient-induced Activation of the Yeast Protein Kinase A (PKA) Target Trehalase

The readdition of an essential nutrient to starved, fermenting cells of the yeast Saccharomyces cerevisiae triggers rapid activation of the protein kinase A (PKA) pathway. Trehalase is activated 5–10-fold within minutes and has been used as a convenient reporter for rapid activation of PKA in vivo. Although trehalase can be phosphorylated and activated by PKA in vitro, demonstration of phosphorylation during nutrient activation in vivo has been lacking. We now show, using phosphospecific antibodies, that glucose and nitrogen activation of trehalase in vivo is associated with phosphorylation of Ser²¹ and Ser⁸³. Unexpectedly, mutants with reduced PKA activity show constitutive phosphorylation despite reduced trehalase activation. The same phenotype was observed upon deletion of the catalytic subunits of yeast protein phosphatase 2A, suggesting that lower PKA activity causes reduced trehalase dephosphorylation. Hence, phosphorylation of trehalase in vivo is not sufficient for activation. Deletion of the inhibitor Dcs1 causes constitutive trehalase activation and phosphorylation. It also enhances binding of trehalase to the 14-3-3 proteins Bmh1 and Bmh2, suggesting that Dcs1 inhibits by preventing 14-3-3 binding. Deletion of Bmh1 and Bmh2 eliminates both trehalase activation and phosphorylation. Our results reveal that trehalase activation in vivo is associated with phosphorylation of typical PKA sites and thus establish the enzyme as a reliable read-out for nutrient activation of PKA in vivo.     

L-Carnosine Affects the Growth of Saccharomyces cerevisiae in a Metabolism-Dependent Manner

The dipeptide L-carnosine (β-alanyl-L-histidine) has been described as enigmatic: it inhibits growth of cancer cells but delays senescence in cultured human fibroblasts and extends the lifespan of male fruit flies. In an attempt to understand these observations, the effects of L-carnosine on the model eukaryote, Saccharomyces cerevisiae, were examined on account of its unique metabolic properties; S. cerevisiae can respire aerobically, but like some tumor cells, it can also exhibit a metabolism in which aerobic respiration is down regulated. L-Carnosine exhibited both inhibitory and stimulatory effects on yeast cells, dependent upon the carbon source in the growth medium. When yeast cells were not reliant on oxidative phosphorylation for energy generation (e.g. when grown on a fermentable carbon source such as 2% glucose), 10–30 mM L-carnosine slowed growth rates in a dose-dependent manner and increased cell death by up to 17%. In contrast, in media containing a non-fermentable carbon source in which yeast are dependent on aerobic respiration (e.g. 2% glycerol), L-carnosine did not provoke cell death. This latter observation was confirmed in the respiratory yeast, Pichia pastoris. Moreover, when deletion strains in the yeast nutrient-sensing pathway were treated with L-carnosine, the cells showed resistance to its inhibitory effects. These findings suggest that L-carnosine affects cells in a metabolism-dependent manner and provide a rationale for its effects on different cell types.     

The G protein-coupled receptor gpr1 is a nutrient sensor that regulates pseudohyphal differentiation in Saccharomyces cerevisiae

Pseudohyphal differentiation in the budding yeast Saccharomyces cerevisiae is induced in diploid cells in response to nitrogen starvation and abundant fermentable carbon source. Filamentous growth requires at least two signaling pathways: the pheromone responsive MAP kinase cascade and the Gpa2p-cAMP-PKA signaling pathway. Recent studies have established a physical and functional link between the Galpha protein Gpa2 and the G protein-coupled receptor homolog Gpr1. We report here that the Gpr1 receptor is required for filamentous and haploid invasive growth and regulates expression of the cell surface flocculin Flo11. Epistasis analysis supports a model in which the Gpr1 receptor regulates pseudohyphal growth via the Gpa2p-cAMP-PKA pathway and independently of both the MAP kinase cascade and the PKA related kinase Sch9. Genetic and physiological studies indicate that the Gpr1 receptor is activated by glucose and other structurally related sugars. Because expression of the GPR1 gene is known to be induced by nitrogen starvation, the Gpr1 receptor may serve as a dual sensor of abundant carbon source (sugar ligand) and nitrogen starvation. In summary, our studies reveal a novel G protein-coupled receptor senses nutrients and regulates the dimorphic transition to filamentous growth via a Galpha protein-cAMP-PKA signal transduction cascade.     

Fermentable sugars and intracellular acidification as specific activators of the RAS-adenylate cyclase signalling pathway in yeast: the relationship to nutrient-induced cell cycle control

The RAS proteins of the yeast Saccharomyces cerevisiae fulfil a similar control function on yeast adenylate cyclase as the mammalian Gs proteins on mammalian adenylate cyclase. The discovery that glucose and other fermentable sugars act as specific activators of the RAS-adenylate cyclase pathway in yeast appeared to offer a mechanism for the way in which at least one nutrient would control progression over the start point in the G1 phase of the yeast cell cycle by means of this pathway. Recently, however, evidence has been obtained to show that the glucose-activation pathway of adenylate cyclase is a glucose-repressible pathway and therefore not operative during growth on glucose. In addition, mutant strains were obtained which lack the glucose-activation pathway and show normal exponential growth on glucose. This appears to confine the physiological role of this pathway to control of the transition from the derepressed state (growth on respirative carbon sources) to the repressed state (growth on fermentative carbon sources) by means of an already well-documented cAMP-triggered protein phosphorylation cascade. Intracellular acidification also stimulates the RAS-adenylate cyclase pathway, which might constitute a rescue mechanism for cells suffering from stress conditions. The presence of a nitrogen source does not stimulate the RAS-adenylate cyclase pathway. Although other nutrient signals for the pathway might still be discovered, it appears more and more likely that the well-known requirement of cAMP for progression over the start point of the yeast cell cycle is limited to providing a basal cAMP level rather than acting as a second messenger for an extracellular signal.(ABSTRACT TRUNCATED AT 250 WORDS)     

A theoretical and empirical investigation of delayed growth response in the continuous culture of bacteria

When the growth of bacteria in a chemostat is controlled by limiting the supply of a single essential nutrient, the growth rate is affected both by the concentration of this nutrient in the culture medium and by the amount of time that it takes for the chemical and physiological processes that result in the production of new biomass. Thus, although the uptake of nutrient by cells is an essentially instantaneous process, the addition of new biomass is delayed by the amount of time that it takes to metabolize the nutrient. Mathematical models that incorporate this "delayed growth response" (DGR) phenomenon have been developed and analysed. However, because they are formulated in terms of parameters that are difficult to measure directly, these models are of limited value to experimentalists. In this paper, we introduce a DGR model that is formulated in terms of measurable parameters. In addition, we provide for this model a complete set of criteria for determining persistence versus extinction of the bacterial culture in the chemostat. Specifically, we show that DGR plays a role in determining persistence versus extinction only under certain ranges of chemostat operating parameters. It is also shown, however, that DGR plays a role in determining the steady-state nutrient and bacteria concentrations in all instances of persistence. The steady state and transient behavior of solutions of our model is found to be in agreement with data that we obtained in growing Escherichia coli 23716 in a chemostat with glucose as a limiting nutrient. One of the theoretical predictions of our model that does not occur in other DGR models is that under certain conditions a large delay in growth response might actually have a positive effect on the bacteria's ability to persist.     

On-chip single-cell microcultivation assay for monitoring environmental effects on isolated cells

We have developed a on-chip single-cell microcultivation assay as a means of observing the adaptation process of single bacterial cells during nutrient concentration changes. This assay enables the direct observation of single cells captured in microchambers made on thin glass slides and having semipermeable membrane lids, in which cells were kept isolated with optical tweezers. After changing a medium of 0.2% (w/v) glucose concentration to make it nutrient-free 0.9% NaCl medium, the growth of all cells inserted into the medium stopped within 20 min, irrespective of their cell cycles. When a nutrient-rich medium was restored, the cells started to grow again, even after the medium had remained nutrient-free for 42 h. The results indicate that the cell's growth and division are directly related to their nutrient condition. The growth curve also indicates that the cells keep their memory of what their growth and division had been before they stopped growing.     

The Saccharomyces cerevisiae alcohol acetyl transferase gene ATF1 is a target of the cAMP/PKA and FGM nutrient-signalling pathways

The ATF1-encoded Saccharomyces cerevisiae yeast alcohol acetyl transferase I is responsible for the formation of several different volatile acetate esters during fermentations. A number of these volatile esters, e.g. ethyl acetate and isoamyl acetate, are amongst the most important aroma compounds in fermented beverages such as beer and wine. Manipulation of the expression levels of ATF1 in brewing yeast strains has a significant effect on the ester profile of beer. Northern blot analysis of ATF1 and its closely related homologue, Lg-ATF1, showed that these genes were rapidly induced by the addition of glucose to anaerobically grown carbon-starved cells. This induction was abolished in a protein kinase A (PKA)-attenuated strain, while a PKA-overactive strain showed stronger ATF1 expression, indicating that the Ras/cAMP/PKA signalling pathway is involved in this glucose induction. Furthermore, nitrogen was needed in the growth medium in order to maintain ATF1 expression. Long-term activation of ATF1 could also be obtained by the addition of the non-metabolisable amino acid homologue beta-L-alanine, showing that the effect of the nitrogen source did not depend on its metabolism. In addition to nutrient regulation, ATF1 and Lg-ATF1 expression levels were also affected by heat and ethanol stress. These findings help in the understanding of the effect of medium composition on volatile ester synthesis in industrial fermentations. In addition, the complex regulation provides new insights into the physiological role of Atf1p in yeast.     

Cytosolic pH is a second messenger for glucose and regulates the PKA pathway through V‐ATPase

Glucose is the preferred carbon source for most cell types and a major determinant of cell growth. In yeast and certain mammalian cells, glucose activates the cAMP‐dependent protein kinase A (PKA), but the mechanisms of PKA activation remain unknown. Here, we identify cytosolic pH as a second messenger for glucose that mediates activation of the PKA pathway in yeast. We find that cytosolic pH is rapidly and reversibly regulated by glucose metabolism and identify the vacuolar ATPase (V‐ATPase), a proton pump required for the acidification of vacuoles, as a sensor of cytosolic pH. V‐ATPase assembly is regulated by cytosolic pH and is required for full activation of the PKA pathway in response to glucose, suggesting that it mediates, at least in part, the pH signal to PKA. Finally, V‐ATPase is also regulated by glucose in the Min6 β‐cell line and contributes to PKA activation and insulin secretion. Thus, these data suggest a novel and potentially conserved glucose‐sensing pathway and identify a mechanism how cytosolic pH can act as a signal to promote cell growth.     

Cell volume increase in Escherichia coli after shifts to richer media.

Synchronous cultures of Escherichia coli 15-THU and WP2s, which were selected by velocity sedimentation from exponential-phase cultures growing in an acetate-minimal salts medium, were shifted to richer media at various times during the cell cycle by the addition of glucose or nutrient broth. Cell numbers and mean cell volumes were measured electronically. The duration of the division cycle of the shifted generation was not altered significantly by the addition of either nutrient. Growth rates, measured as rates of cell volume increase, were constant throughout the cycle in unshifted acetate control cultures. When glucose was added, growth rates also remained unchanged during the remainder of the cell cycle and then increased abruptly at or after cell division. When nutrient broth was added, growth rates remained unchanged from periods of 0.2 to 0.4 generations and then increased abruptly to their final values. In all cases, the cell volume increase was linear both before and after the growth rate transition. The strongest support for a linear cell volume increase during the cell cycle of E. coli in slowly growing acetate cultures, however, was obtained in unshifted cultures, in complete agreement with earlier observations of cell volumes at much more rapid growth rates. Although cell growth and division are under the control of the synthesizing machinery in the cell responsible for RNA and protein synthesis, the results indicate that growth is also regulated by membrane-associated transport systems.     

Roles of phosphatidylethanolamine and of its several biosynthetic pathways in Saccharomyces cerevisiae

Three different pathways lead to the synthesis of phosphatidylethanolamine (PtdEtn) in yeast, one of which is localized to the inner mitochondrial membrane. To study the contribution of each of these pathways, we constructed a series of deletion mutants in which different combinations of the pathways are blocked. Analysis of their growth phenotypes revealed that a minimal level of PtdEtn is essential for growth. On fermentable carbon sources such as glucose, endogenous ethanolaminephosphate provided by sphingolipid catabolism is sufficient to allow synthesis of the essential amount of PtdEtn through the cytidyldiphosphate (CDP)-ethanolamine pathway. On nonfermentable carbon sources, however, a higher level of PtdEtn is required for growth, and the amounts of PtdEtn produced through the CDP-ethanolamine pathway and by extramitochondrial phosphatidylserine decarboxylase 2 are not sufficient to maintain growth unless the action of the former pathway is enhanced by supplementing the growth medium with ethanolamine. Thus, in the absence of such supplementation, production of PtdEtn by mitochondrial phosphatidylserine decarboxylase 1 becomes essential. In psd1Delta strains or cho1Delta strains (defective in phosphatidylserine synthesis), which contain decreased amounts of PtdEtn, the growth rate on nonfermentable carbon sources correlates with the content of PtdEtn in mitochondria, suggesting that import of PtdEtn into this organelle becomes growth limiting. Although morphological and biochemical analysis revealed no obvious defects of PtdEtn-depleted mitochondria, the mutants exhibited an enhanced formation of respiration-deficient cells. Synthesis of glycosylphosphatidylinositol-anchored proteins is also impaired in PtdEtn-depleted cells, as demonstrated by delayed maturation of Gas1p. Carboxypeptidase Y and invertase, on the other hand, were processed with wild-type kinetics. Thus, PtdEtn depletion does not affect protein secretion in general, suggesting that high levels of nonbilayer-forming lipids such as PtdEtn are not essential for membrane vesicle fusion processes in vivo.     

SLK1, a yeast homolog of MAP kinase activators, has a RAS/cAMP-independent role in nutrient sensing

The Saccharomyces cerevisiae SLK1 protein is implicated in nutrient sensing and growth control. Under nutrient-limiting conditions, slk1 mutants fail to undergo cell cycle arrest. The role of the SLK1 protein in nutrient sensing was examined with respect to the cAMP-dependent protein kinase (PKA) pathway, which has a well characterized role in growth control in yeast, and by the analysis of dominant SLK1 alleles that affect the nutrient response of wild-type cells. Interactions with the PKA pathway were examined by phenotypic analysis of double mutants of slk1 and various PKA pathway mutants. Combining the slk1- mutation with a mutation that is thought constitutively activate the PKA pathway, pde2, resulted in enhanced growth control defects. The combination of slk1- with mutations that inhibit the PKA pathway, cdc25 and ras1 ras2, failed to alleviate the slk1 cell cycle arrest defect and lowered the permissive temperature for growth. Furthermore bcy1 tpk1 tpk2 tpk3 ^w (bcyl tpk ^w) mutants, which have constitutive, low-level, cAMP-independent kinase activity, exhibit nutrient sensing, which is eliminated in the slk1 bcy1 tpk ^w mutants. These results implicated SLK1 in PKA-independent growth control in yeast. The amino-terminal, noncatalytic region of the SLK1 protein may be important in the regulation of SLK1 function in growth control. Overexpression of this region caused starvation sensitivity in wild-type cells by interfering with SLK1 protein function.