Diauxic Shift Research Articles

The target of rapamycin signaling pathway regulates mRNA turnover in the yeast Saccharomyces cerevisiae.

The target of rapamycin (TOR) signaling pathway is an important mechanism by which cell growth is regulated by nutrient availability in eukaryotes. We provide evidence that the TOR signaling pathway controls mRNA turnover in Saccharomyces cerevisiae. During nutrient limitation (diauxic shift) or after treatment with rapamycin (a specific inhibitor of TOR), multiple mRNAs were destabilized, whereas the decay of other mRNAs was unaffected. Our findings suggest that the regulation of mRNA decay by the TOR pathway may play a significant role in controlling gene expression in response to nutrient depletion. The inhibition of the TOR pathway accelerated the major mRNA decay mechanism in yeast, the deadenylation-dependent decapping pathway. Of the destabilized mRNAs, two different responses to rapamycin were observed. Some mRNAs were destabilized rapidly, while others were affected only after prolonged exposure. Our data suggest that the mRNAs that respond rapidly are destabilized because they have short poly(A) tails prematurely either as a result of rapid deadenylation or reduced polyadenylation. In contrast, the mRNAs that respond slowly are destabilized by rapid decapping. In summary, the control of mRNA turnover by the TOR pathway is complex in that it specifically regulates the decay of some mRNAs and not others and that it appears to control decay by multiple mechanisms.

Diauxic shift-induced stress resistance against hydroperoxides in Saccharomyces cerevisiae is not an adaptive stress response and does not depend on functional mitochondria.

Respiring Saccharomyces cerevisiae cells grown on a non-fermentable carbon source are intrinsically more resistant to several stresses, including oxidative stress. The mechanisms leading to increased stress resistance are not yet well understood. Active mitochondria are the major source of intracellular reactive oxygen species (ROS), which could cause the up-regulation of the antioxidant defense systems. We investigated the role of mitochondria in the intrinsic stress resistance against the hydroperoxides H2O2 and tert-butylhydroperoxide 4 h after a shift in carbon source. We found that, independently of functional mitochondria, the yeast acquired the intrinsic resistance of respiring cells against hydroperoxides solely as a response to a change of carbon source in the growth medium. Furthermore, utilizing reporter gene fusion constructs, we monitored the expression of the gamma-glutamylcysteinyl synthetase (encoded by GSH1) and the two superoxide dismutases (encoded by SOD1 and SOD2) during the metabolic transition from fermentation to respiration; and we detected an up-regulation of all three genes during the diauxic shift. Overall available data allowed us to propose that the antioxidant system of S. cerevisiae could be considered as a class of genes under glucose/carbon catabolite regulation. This control system is different from the well-known adaptive response to oxidative stress.

Physiological properties of Saccharomyces cerevisiae from which hexokinase II has been deleted.

Hexokinase II is an enzyme central to glucose metabolism and glucose repression in the yeast Saccharomyces cerevisiae. Deletion of HXK2, the gene which encodes hexokinase II, dramatically changed the physiology of S. cerevisiae. The hxk2-null mutant strain displayed fully oxidative growth at high glucose concentrations in early exponential batch cultures, resulting in an initial absence of fermentative products such as ethanol, a postponed and shortened diauxic shift, and higher biomass yields. Several intracellular changes were associated with the deletion of hexokinase II. The hxk2 mutant had a higher mitochondrial H(+)-ATPase activity and a lower pyruvate decarboxylase activity, which coincided with an intracellular accumulation of pyruvate in the hxk2 mutant. The concentrations of adenine nucleotides, glucose-6-phosphate, and fructose-6-phosphate are comparable in the wild type and the hxk2 mutant. In contrast, the concentration of fructose-1,6-bisphosphate, an allosteric activator of pyruvate kinase, is clearly lower in the hxk2 mutant than in the wild type. The results suggest a redirection of carbon flux in the hxk2 mutant to the production of biomass as a consequence of reduced glucose repression.

Energy-dependent Flip of Fluorescence-labeled Phospholipids Is Regulated by Nutrient Starvation and Transcription Factors, PDR1 and PDR3

The yeast Saccharomyces cerevisiae readily accumulates short-chain, fluorescent 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled phosphatidylcholine and phosphatidylethanolamine at the nuclear envelope/endoplasmic reticulum and mitochondria. The net intracellular accumulation reflects the sum of their inwardly and outwardly directed transbilayer translocation across the plasma membrane (flip and flop, respectively). The rate of flop is negligible in energy-depleted cells as well as at low temperature (2 degrees C). Although flip is reduced at 2 degrees C, it can still be measured by flow cytometry, allowing the rate of flip, independent of flop, to be characterized at this temperature. Flip requires the energy of the plasma membrane proton electrochemical gradient and is down-regulated as cells pass through the diauxic shift and enter stationary phase. Furthermore, drug-resistant, gain-of-function mutations in the transcription factors, PDR1 and PDR3, result in a dramatic down-regulation of flip in addition to their already established up-regulation of flop. These results imply that down-regulation of the NBD-phospholipid flip pathway is a physiological response to environmental stress.

The Transcriptional Activator Cat8p Provides a Major Contribution to the Reprogramming of Carbon Metabolism during the Diauxic Shift in Saccharomyces cerevisiae

In yeast, the transition between the fermentative and the oxidative metabolism, called the diauxic shift, is associated with major changes in gene expression and protein synthesis. The zinc cluster protein Cat8p is required for the derepression of nine genes under nonfermentative growth conditions (ACS1, FBP1, ICL1, IDP2, JEN1, MLS1, PCK1, SFC1, and SIP4). To investigate whether the transcriptional control mediated by Cat8p can be extended to other genes and whether this control is the main control for the changes in the synthesis of the respective proteins during the adaptation to growth on ethanol, we analyzed the transcriptome and the proteome of a cat8 Delta strain during the diauxic shift. In this report, we demonstrate that, in addition to the nine genes known as Cat8p-dependent, there are 25 other genes or open reading frames whose expression at the diauxic shift is altered in the absence of Cat8p. For all of the genes characterized here, the Cat8p-dependent control results in a parallel alteration in mRNA and protein synthesis. It appears that the biochemical functions of the proteins encoded by Cat8p-dependent genes are essentially related to the first steps of ethanol utilization, the glyoxylate cycle, and gluconeogenesis. Interestingly, no function involved in the tricarboxylic cycle and the oxidative phosphorylation seems to be controlled by Cat8p.

Integrating naive Bayes models and external knowledge to examine copper and iron homeostasis in S. cerevisiae.

A novel suite of analytical techniques and visualization tools are applied to 78 published transcription profiling experiments monitoring 5,687 Saccharomyces cerevisiae genes in studies examining cell cycle, responses to stress, and diauxic shift. A naive Bayes model discovered and characterized 45 classes of gene profile vectors. An enrichment measure quantified the association between these classes and specific external knowledge defined by four sets of categories to which genes can be assigned: 106 protein functions, 5 stages of the cell cycle, 265 transcription factors, and 16 chromosomal locations. Many of the 38 genes in class 42 are known to play roles in copper and iron homeostasis. The 17 uncharacterized open reading frames in this class may be involved in similar homeostatic processes; human homologs of two of them could be associated with as yet undefined disease states arising from aberrant metal ion regulation. The Met4, Met31, and Met32 transcription factors may play a role in coregulating genes involved in copper and iron metabolism. Extensions of the simple graphical model used for clustering to learning more complex models of genetic networks are discussed.

Glucose depletion causes haploid invasive growth in yeast.

Haploid yeast invades solid agar in response to nutrient limitation. To decipher the cues that underlie invasion, we have developed a single cell invasive growth assay. Using this assay, as well as the traditional plate-washing assay, we show that invasive growth occurs in response to glucose depletion. In the absence of glucose (or other fermentable sugar), individual cells adopted a nonaxial budding pattern and elongated morphology within the first cell divisions, and invasion into the agar was observed in microcolonies containing as few as 10 cells. In support of this observation, we found that glucose suppressed the hyperinvasive growth morphology of STE11-4, pbs2, hsl7, and RAS2V19 mutations. In addition, removal of glucose from YPD medium caused constitutive invasion in wild-type cells. We tested glucose control proteins for a role in invasion and found that Snf1, a protein required for derepression of glucose-repressed genes, was required for invasive growth. The transcription factor Sip4, which interacts with Snf1 and is induced during the diauxic shift, had an inhibitory role on invasive growth, suggesting that multiple mechanisms are required for glucose depletion-dependent invasion.

Hyperphosphorylation of Msn2p and Msn4p in response to heat shock and the diauxic shift is inhibited by cAMP in Saccharomyces cerevisiae.

In response to various stresses, as well as during the diauxic transition, the Msn2p and Msn4p transcription factors of Saccharomyces cerevisiae are activated and induce a large set of genes. This activation is inhibited by the Ras/cAMP/PKA (cAMP-dependent protein kinase) pathway. Here we show by immunoblotting experiments that Msn2p and Msn4p are phosphorylated in vivo during growth on glucose, and become hyperphosphorylated at the diauxic transition and upon heat shock. This hyperphosphorylation is correlated with activation of Msn2/4p-dependent transcription. An increased level of cAMP prevents and reverses these hyperphosphorylations, indicating that kinases other than PKA are involved. These results suggest that PKA and stress-activated kinases control Msn2/4p activity by antagonistic phosphorylation. It was also noted that Msn4p is transiently increased at the diauxic transition. Msn2p and Msn4p present different hyperphosphorylation patterns in response to different stresses.

The NC2 repressor is dispensable in yeast mutated for the Sin4p component of the holoenzyme and plays roles similar to Mot1p in vivo

NC2 (Dr1/DRAP1) and Mot1p are global repressors of transcription that have been isolated in both Saccharomyces cerevisiae and humans. NC2 is a dimeric histone-fold complex that represses RNA polymerase II transcription through binding to TBP and inhibition of TFIIA and TFIIB. Mot1p is an ATPase that removes DNA-bound TBP upon ATP hydrolysis. In this work, we studied the core promoter specificity of NC2 in vivo using a strain that carries mutated NC2beta activity. We show that NC2, like Mot1p, is required for transcription of the HIS3 and HIS4 TATA-less core promoters. Furthermore, whereas neither Mot1p nor NC2 appear to function as repressors of the HIS3 gene in cells growing exponentially in glucose, we find that both are required for repression of the HIS3 TATA promoter when cells go through the diauxic shift. Thus, the activity of these factors is similarly regulated depending upon the physiological conditions, and it appears that core promoters activated or repressed by them in vivo might be distinguishable by whether or not they contain a canonical TATA sequence. Finally, although NC2 is an essential factor for yeast viability, we isolated a mutation in a non-essential component of the holoenzyme, Sin4p, that bypasses the requirement for NC2.

Complex evolution of the inositol-1-phosphate synthase gene among archaea and eubacteria

have a correlation of 20.15 (there are many similar examples). In certain cases, the patterns can be different owing to differences in mRNA stability ‐ for example, the same amount of mRNA might be transcribed, but differential degradation rates could affect the expression measurements of the microarrays. However, in the many examples of completely different expression pattern pairs, it is more likely that separate regulatory systems control the genes. Considering the gene pairs YBR189W‐YBR191W, YJL190C‐YJL189W and YOL040C‐YOL039W in more detail, the first pair is transcribed on the same strand, whereas the other two pairs are transcribed away from each other on opposite strands. In the first case, the regulatory system of both genes could be located upstream of YBR189W. In the other cases, it might be located between the genes. The expression patterns from the cell cycle data are shown in Fig. 2b‐d. These genes code ribosomal protein, and as the genes are functionally related, it is not surprising that all six patterns are similar. However, the expression patterns for the genes in each pair are almost identical. The average correlation between adjacent genes is 0.95, whereas the average correlation between the groups is 0.85. In the diauxic shift data, the corresponding correlations are 0.96 and 0.93. The values are 0.83 and 0.67 in the sporulation data. The correlation values support the notion that the consecutive genes are controlled by a single regulatory system. Many genes that are controlled by separate regulatory systems might have highly correlated expression patterns. This could be the case if the genes are functionally related, or it might occur by random chance. If genes are controlled by a single regulatory system, then their expression patterns should be highly correlated in any data set. Each data set that becomes available can be used to generate a list of adjacent genes with highly correlated expression patterns. By intersecting these lists we obtain the most likely candidates for control by a single regulatory system. Table 1 presents some of the best candidates based on the three expression data sets. A complete candidate list can be found at http://www-hto.usc.edu/~tanghx/yeast-gene-pair.html, and code for conducting searches in other data sets is available from the authors. Once strong candidates have been found, the issue can be resolved by experimental analysis of yeast strains having a mutation in the putative site of the single regulatory region. If both genes are affected, then they are controlled by a single regulatory system.

Stress response and expression patterns in wine fermentations of yeast genes induced at the diauxic shift.

During wine fermentation yeasts quickly reach a stationary phase, where cells are metabolically active by consuming sugars present in grape must. It is, consequently, of great interest at this stage to identify suitable gene promoters that may be used to induce the expression of genes with enological applications. With this aim, we have studied a group of genes showing an induction peak at the diauxic shift, and possessing stress response elements (STRE) at their promoters. We have determined their induction levels under individualized stress conditions, such as carbon source starvation or high salt concentrations. In all the cases studied, the activation and/or basal transcription are dependent on the transcriptional factors Msn2p and Msn4p. We have analysed the expression patterns and mRNA levels during wine fermentation, and have found that they are all activated at the stationary phase. Finally, we have identified SPI1, a new highly expressed yeast gene which is specifically induced at the stationary phase of both microvinification and laboratory growth conditions.

Stress response and expression patterns in wine fermentations of yeast genes induced at the diauxic shift

Stress response and expression patterns in wine fermentations of yeast genes induced at the diauxic shift

Rapamycin-modulated transcription defines the subset of nutrient-sensitive signaling pathways directly controlled by the Tor proteins.

The immunosuppressant rapamycin inhibits Tor1p and Tor2p (target of rapamycin proteins), ultimately resulting in cellular responses characteristic of nutrient deprivation through a mechanism involving translational arrest. We measured the immediate transcriptional response of yeast grown in rich media and treated with rapamycin to investigate the direct effects of Tor proteins on nutrient-sensitive signaling pathways. The results suggest that Tor proteins directly modulate the glucose activation and nitrogen discrimination pathways and the pathways that respond to the diauxic shift (including glycolysis and the citric acid cycle). Tor proteins do not directly modulate the general amino acid control, nitrogen starvation, or sporulation (in diploid cells) pathways. Poor nitrogen quality activates the nitrogen discrimination pathway, which is controlled by the complex of the transcriptional repressor Ure2p and activator Gln3p. Inhibiting Tor proteins with rapamycin increases the electrophoretic mobility of Ure2p. The work presented here illustrates the coordinated use of genome-based and biochemical approaches to delineate a cellular pathway modulated by the protein target of a small molecule.

Yeast Carboxyl-terminal Domain Kinase I Positively and Negatively Regulates RNA Polymerase II Carboxyl-terminal Domain Phosphorylation

Monoclonal antibodies that recognize specific carboxyl-terminal domain (CTD) phosphoepitopes were used to examine CTD phosphorylation in yeast cells lacking carboxyl-terminal domain kinase I (CTDK-I). We show that deletion of the kinase subunit CTK1 results in an increase in phosphorylation of serine in position 5 (Ser(5)) of the CTD repeat (Tyr(1)-Ser(2)-Pro(3)-Thr(4)-Ser(5)-Pro(6)-Ser(7)) during logarithmic growth. This result indicates that CTDK-I negatively regulates CTD Ser(5) phosphorylation. We also show that CTK1 deletion (ctk1Delta) eliminates the transient increase in CTD serine 2 (Ser(2)) phosphorylation observed during the diauxic shift. This result suggests that CTDK-I may play a direct role in phosphorylating CTD Ser(2) in response to nutrient depletion. Northern blot analysis was used to show that genes normally induced during the diauxic shift are not properly induced in a ctk1Delta strain. Glycogen synthase (GSY2) and cytosolic catalase (CTT1) mRNA levels increase about 10-fold in wild-type cells, but this increase is not observed in ctk1Delta cells suggesting that increased message levels may require Ser(2) phosphorylation. Heat shock also induces Ser(2) phosphorylation, but we show here that this change in CTD modification and an accompanying induction of heat shock gene expression is independent of CTDK-I. The observation that SSA3/SSA4 expression is increased in ctk1Delta cells grown at normal temperature suggests a possible role for CTDK-I in transcription repression. We discuss several possible positive and negative roles for CTDK-I in regulating CTD phosphorylation and gene expression.

Dynamic responses of reserve carbohydrate metabolism under carbon and nitrogen limitations in Saccharomyces cerevisiae.

The dynamic responses of reserve carbohydrates with respect to shortage of either carbon or nitrogen source was studied to obtain a sound basis for further investigations devoted to the characterization of mechanisms by which the yeast Saccharomyces cerevisiae can cope with nutrient limitation during growth. This study was carried out in well-controlled bioreactors which allow accurate monitoring of growth and frequent sampling without disturbing the culture. Under glucose limitation, genes involved in glycogen and trehalose biosynthesis (GLG1, GSY1, GSY2, GAC1, GLC3, TPS1), in their degradation (GPH1, NTHI), and the typical stress-responsive CTT1 gene were coordinately induced in parallel with glycogen, when the growth has left the pure exponential phase and while glucose was still plentiful in the medium. Trehalose accumulation was delayed until the diauxic shift, although TPS1 was induced much earlier, due to hydrolysis of trehalose by high trehalase activity. In contrast, under nitrogen limitation, both glycogen and trehalose began to accumulate at the precise time when the nitrogen source was exhausted from the medium, coincidentally with the transcriptional activation of genes involved in their metabolism. While this response to nitrogen starvation was likely mediated by the stress-responsive elements (STREs) in the promoter of these genes, we found that these elements were not responsible for the co-induction of genes involved in reserve carbohydrate metabolism during glucose limitation, since GLG1, which does not contain any STRE, was coordinately induced with GSY2 and TPS1.

The general stress sigma factor sigmaS of Escherichia coli is induced during diauxic shift from glucose to lactose.

The general stress sigma factor sigmaS (RpoS) of Escherichia coli is strongly induced in response to glucose starvation. This increase in the cellular sigmaS level is due to stabilization of sigmaS, which under non-stress conditions is subject to rapid proteolysis. In the present study, it is demonstrated that sigmaS is also induced during the diauxic shift from glucose to lactose, i.e., under conditions of glucose exhaustion in the presence of another, less-preferred carbon source that eventually gets utilized. This sigmaS induction, which is due to stabilization, is transient and precedes the induction of beta-galactosidase. In parallel, sigmaS-dependent genes are transiently activated, as was shown here for osmY. Although sigmaS can mediate transcription of lacZ in vitro, sigmaS does not contribute to the induction of beta-galactosidase during the diauxic lag phase. Rather, the induction of sigmaS and the general stress response during the diauxic shift plays the role of a rapidly activated emergency system, which is shut off again as soon as the cells are able to cope with the stress situation by utilizing a more specific and more economical system.

A Novel Membrane-bound Glutathione S-Transferase Functions in the Stationary Phase of the Yeast Saccharomyces cerevisiae

The glutathione S-transferases (GSTs) represent a significant group of detoxification enzymes that play an important role in drug resistance in all eukaryotic species. In this paper we report an identification and characterization of the two Saccharomyces cerevisiae genes, GTT1 and GTT2 (glutathione transferase 1 and 2), coding for functional GST enzymes. Despite only limited similarity with GSTs from other organisms (approximately 50%), recombinant Gtt1p and Gtt2p exhibit GST activity with 1-chloro-2, 4-dinitrobenzene as a substrate. Both Gtt1p and Gtt2p are able to form homodimers, as determined by two hybrid assay. Subcellular fractionation demonstrated that Gtt1p associates with the endoplasmic reticulum. Expression of GTT1 is induced after diauxic shift and remains high throughout the stationary phase. Strains deleted for GTT1 and/or GTT2 are viable but exhibit increased sensitivity to heat shock in stationary phase and limited ability to grow at 39 degreesC.

The highly conserved, coregulated SNO and SNZ gene families in Saccharomyces cerevisiae respond to nutrient limitation.

SNZ1, a member of a highly conserved gene family, was first identified through studies of proteins synthesized in stationary-phase yeast cells. There are three SNZ genes in Saccharomyces cerevisiae, each of which has another highly conserved gene, named SNO (SNZ proximal open reading frame), upstream. The DNA sequences and relative positions of SNZ and SNO genes have been phylogenetically conserved. This report details studies of the expression of the SNZ-SNO gene pairs under various conditions and phenotypic analysis of snz-sno mutants. An analysis of total RNA was used to determine that adjacent SNZ-SNO gene pairs are coregulated. SNZ2/3 and SNO2/3 mRNAs are induced prior to the diauxic shift and decrease in abundance during the postdiauxic phase, when SNZ1 and SNO1 are induced. In snz2 snz3 mutants, SNZ1 mRNA is induced prior to the diauxic shift, when SNZ2/3 mRNAs are normally induced. Under nitrogen-limiting conditions, SNZ1 mRNAs accumulate in tryptophan, adenine, and uracil auxotrophs but not in prototrophic strains, indicating that induction occurs in response to the limitation of specific nutrients. Strains carrying deletions in all SNZ-SNO gene pairs are viable, but snz1 and sno1 mutants are sensitive to 6-azauracil (6-AU), an inhibitor of purine and pyrimidine biosynthetic enzymes, and methylene blue, a producer of singlet oxygen. The conservation of sequence and chromosomal position, the coregulation and pattern of expression of SNZ1 and SNO1 genes, and the sensitivity of snz1 and sno1 mutants to 6-AU support the hypothesis that the associated proteins are part of an ancient response to nutrient limitation.

TheGTS1Gene Product Influences the Ultradian Oscillation of Glycolysis in Cell Suspension of the YeastSaccharomyces cerevisiae

We tested the effect of theGTS1gene of the yeastSaccharomyces cerevisiaeon the cyanide-induced ultradian oscillation of the glycolytic metabolite NADH in cell suspension of strains with different copy numbers of the gene, that is, the wild-type,GTS1-disrupted andGTS1-overexpressing strains. The cells showed long-lasting oscillations when harvested between 1 and 2 hours after the diauxic shift from glucose to ethanol as a growth substrate. The frequencies of oscillation did not vary very much among the three strains tested. However, the amplitudes and durations of the oscillation were changed significantly as a function of theGTS1gene-dosage. The effect of GTS1 on the amplitude was not caused by changing rates of glucose incorporation into cells as the rates were the same among the three strains during the macroscopic oscillation.

Msn2p and Msn4p control a large number of genes induced at the diauxic transition which are repressed by cyclic AMP in Saccharomyces cerevisiae.

The multicopy suppressors of the snf1 defect, Msn2p and Msn4p transcription factors (Msn2/4p), activate genes through the stress-responsive cis element (CCCCT) in response to various stresses. This cis element is also the target for repression by the cyclic AMP (cAMP)-signaling pathway. We analyzed the two-dimensional gel electrophoresis pattern of protein synthesis of the msn2 msn4 double mutant and compared it with that of the wild-type strain during exponential growth phase and at the diauxic transition. Thirty-nine gene products (including those of ALD3, GDH3, GLK1, GPP2, HSP104, HXK1, PGM2, SOD2, SSA3, SSA4, TKL2, TPS1, and YBR149W) are dependent upon Msn2/4p for their induction at the diauxic transition. The expression of all these genes is repressed by cAMP. Thirty other genes identified during this study are still inducible in the mutant. A subset of these genes were found to be superinduced at the diauxic transition, and others were subject to cAMP repression (including ACH1, ADH2, ALD6, ATP2, GPD1, ICL1, and KGD2). We conclude from this analysis that Msn2/4p control a large number of genes induced at the diauxic transition but that other, as-yet-uncharacterized regulators, also contribute to this response. In addition, we show here that cAMP repression applies to both Msn2/4p-dependent and -independent control of gene expression at the diauxic shift. Furthermore, the fact that all the Msn2/4p gene targets are subject to cAMP repression suggests that these regulators could be targets for the cAMP-signaling pathway.